Bispecific antibody exhibiting increased alternative fviii-cofactor-function activity

ABSTRACT

The present invention provides light chain amino acid substitutions that improve the FVIII cofactor function-substituting activity of ACE910 (Emicizumab), novel light chains showing FVIII cofactor function-substituting activity, and heavy chain amino acid substitutions that improve the FVIII cofactor function-substituting activity of novel light chain-containing bispecific antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International Application Serial No. PCT/JP2017/027152, filed on Jul. 27, 2017, which claims the benefit of Japanese Application Serial No. 2016-150769, filed on Jul. 29, 2016.

TECHNICAL FIELD

The present invention relates to antibodies having enhanced FVIII cofactor function-substituting activity, pharmaceutical formulations comprising such antibodies as active ingredients, methods for producing such antibodies, and such. More specifically, the present invention relates to bispecific antibodies having FVIII cofactor function-substituting activity higher than that of ACE910 (Emicizumab).

BACKGROUND ART

Hemophilia A is a bleeding abnormality caused by a hereditary decrease or deficiency of blood coagulation factor VIII (FVIII) function. Hemophilia A patients are generally administered with an FVIII formulation for the bleeding (on-demand administration). In recent years, FVIII formulations are administered prophylactically to prevent bleeding events (NPLs 1 and 2) (preventive administration). The blood half-life of FVIII formulations is approximately 12 hours to 16 hours. Therefore, for continuous prevention, FVIII formulations are administered to patients three times a week (NPLs 3 and 4). In on-demand administrations, FVIII formulations are also additionally administered when necessary at regular intervals to prevent rebleeding. In addition, FVIII formulations are administered intravenously. Therefore, there has been a strong need for pharmaceutical agents with lesser burden of administration than FVIII formulations.

Antibodies against FVIII (inhibitors) occasionally develop in hemophilia patients. Such inhibitors counteract the effects of the FVIII formulations. For bleeding in patients who have developed inhibitors (inhibitor patients), bypassing formulations are administered. Their mechanisms of action are not dependent on FVIII function, that is, the function of catalyzing the activation of blood coagulation factor X (FX) by activated blood coagulation factor IX (FIXa). Therefore, in some cases, bypassing formulations cannot sufficiently stop the bleeding. Accordingly, there has been a strong need for pharmaceutical agents that are not affected by the presence of inhibitors and which can substitute for FVIII function.

As a means for solving these problems, bispecific antibodies that substitute for FVIII function and their uses have been reported (PTLs 1, 2, 3, and 4). The bispecific antibodies against FIXa and FX can substitute for FVIII function and exhibit FVIII cofactor function-substituting activity by positioning the two factors in proximity (NPL 5). The FVIII cofactor function-substituting activity of the bispecific antibodies against FIXa and FX refers to the activity calculated from examination of FX activation reaction by FIXa using colorimetric determination methods and a thrombin generation assay using hemophilia A plasma. It has been reported that the FVIII cofactor function-substituting activity of the antibodies can be enhanced by optimizing the affinity towards FIXa and FX (NPL 6). Furthermore, the FVIII cofactor function-substituting activity of the antibodies is known to be affected by the IgG isotype, disulfide bond pattern, amino acid sequence of the hinge region, and the presence or absence of sugar chains in the Fc region (NPL 7). ACE910 (Emicizumab), which is one of these antibodies and has high FVIII cofactor function-substituting activity, has been reported to exhibit hemostatic effects in monkey models of hemophilia (NPLs 8 and 9). Furthermore, in clinical trials of ACE910 (Emicizumab) on healthy subjects, excellent pharmacokinetics (long half-life) and tolerability have been confirmed (NPL 10), and in clinical trials on hemophilia A patients with or without inhibitors, the number of bleeding episodes was remarkably suppressed by ACE910 (Emicizumab) administration compared to before ACE910 (Emicizumab) administration (NPL 11).

As described, suppressive effects on the number of bleeding episodes have been observed for ACE910 (Emicizumab) in clinical trials. However, since improvement effects by ACE910 (Emicizumab) on the maximum amount of thrombin generation (peak height) in in vitro thrombin generation assays using FVIII-deficient plasma were lower than the normal level of FVIII activity which is 100 U/dL (NPL 8), further enhancement of drug efficacy has been desired. In addition, bispecific antibodies which can further reduce the administered dose through improvement of specific activity, and such, have been desired.

ACE910 (Emicizumab) is a bispecific antibody having high FVIII cofactor function-substituting activity, which was prepared as follows: anti-FIX antibody and/or anti-FIXa antibody and anti-FX antibody acquired by animal immunization were humanized to obtain hBS1 as a lead antibody; and many amino acid substitutions were introduced into the lead antibody to multilaterally optimize it (NPL 6 and PTL 4). For enhancement of drug efficacy and improvement of specific activity, there is a need for a bispecific antibody that substitutes for FVIII function, which has higher maximum activity (maximum FVIII cofactor function-substituting activity) than ACE910 (Emicizumab) and can also exhibit FVIII cofactor function-substituting activity at concentrations lower than that of ACE910 (Emicizumab). However, to date, there are no reports of bispecific antibodies having FVIII cofactor function-substituting activity higher than that of ACE910 (Emicizumab) from the viewpoint of concentration and maximum activity.

CITATION LIST Patent Literature

-   [PTL 1] WO 2005/035754 -   [PTL 2] WO 2005/035756 -   [PTL 3] WO 2006/109592 -   [PTL 4] WO 2012/067176

Non-patent Literature

-   [NPL 1] Blood 58, 1-13 (1981) -   [NPL 2] Nature 312, 330-337 (1984) -   [NPL 3] Nature 312, 337-342 (1984) -   [NPL 4] Biochim. Biophys. Acta 871, 268-278 (1986) -   [NPL 5] Nat Med. 2012 October; 18(10): 1570-4. -   [NPL 6] PLoS One. 2013; 8(2): e57479. -   [NPL 7] MAbs. 2015; 7(1):120-8. -   [NPL 8] J Thromb Haemost. 2014 February; 12(2): 206-213. -   [NPL 9] Blood. 2014 Nov. 13; 124(20): 3165-71. -   [NPL 10] Blood. 2016 Mar. 31; 127(13): 1633-1641 -   [NPL 11] New Eng J Med 2016 May 26; 374(21): 2044-2053

SUMMARY OF INVENTION Problems to be Solved by the Invention

The present invention was achieved in view of the above circumstances. An objective of the present invention is to provide antibodies having increased FVIII cofactor function-substituting activity, pharmaceutical formulations comprising such antibodies as active ingredients, and methods of producing them. More specifically, an objective of the present invention is to provide mutations in the heavy-chain and light-chain variable region sites or novel light chain CDR sequences that are different from those of ACE910 (Emicizumab) for preparing bispecific antibodies having FVIII cofactor function-substituting activity higher than that of ACE910 (Emicizumab), bispecific antibodies which have such mutations or such CDR sequences and recognize FIX and/or FIXa, and FX, methods of producing such antibodies, pharmaceutical formulations comprising such antibodies as active ingredients, and methods for treating hemophilia A using such pharmaceutical formulations.

Means for Solving the Problems

To solve the above-mentioned problems, the present inventors produced variants of ACE910 (Emicizumab) by introducing amino acid substitutions into various sites of its light-chain variable regions and succeeded in discovering amino acid substitutions that enhance the FVIII cofactor function-substituting activity. The inventors also succeeded in obtaining from human antibody libraries, novel light chains having sequences different from those of ACE910, which have FVIII cofactor function-substituting activity, and in identifying amino acid substitutions in the light chains, which enhance the FVIII cofactor function-substituting activity.

Furthermore, when variants of bispecific antibodies prepared by using these light chains were produced by introducing amino acid substitutions into various sites in the heavy chain variable regions, the inventors succeeded in discovering amino acid substitutions that enhance the FVIII cofactor function-substituting activity. The present invention is based on such findings, and specifically provides the following:

-   [1] a polypeptide comprising an antibody light chain variable     domain, wherein the polypeptide comprises an antibody light chain     variable domain having the light chain CDR 1, 2, and 3 amino acid     sequences of SEQ ID NOs: 7, 8, and 9, respectively, or an antibody     light chain variable domain having the amino acid sequence of SEQ ID     NO: 47, in which one or more amino acids selected from the group     consisting of K24, A25, S26, R27, N28, 129, E30, R31, Q32, L33, A34,     Q50, A51, S52, R53, K54, E55, S56, Q89, Q90, Y91, S92, D93, P94,     P95, L96, and T97, numbered according to the Kabat numbering system,     have been substituted with arbitrary amino acids other than     cysteine; -   [2] a bispecific antibody which recognizes FIX and/or FIXa, and FX,     wherein the antibody comprises an antibody light chain variable     domain having the light chain CDR 1, 2, and 3 amino acid sequences     of SEQ ID NOs: 7, 8, and 9, respectively, or an antibody light chain     variable domain having the amino acid sequence of SEQ ID NO: 47, in     which one or more amino acids selected from the group consisting of     K24, A25, S26, R27, N28, 129, E30, R31, Q32, L33, A34, Q50, A51,     S52, R53, K54, E55, S56, Q89, Q90, Y91, S92, D93, P94, P95, L96, and     T97, numbered according to the Kabat numbering system, have been     substituted with arbitrary amino acids other than cysteine; -   [3] a bispecific antibody which recognizes FIX and/or FIXa, and FX,     wherein a first polypeptide and a third polypeptide form a pair and     a second polypeptide and a fourth polypeptide form a pair, wherein     the first polypeptide comprises the antibody heavy chain variable     domain amino acid sequence of SEQ ID NO: 45 and the second     polypeptide comprises the antibody heavy chain variable domain amino     acid sequence of SEQ ID NO: 46, wherein either the third polypeptide     or the fourth polypeptide comprises the antibody light chain     variable domain amino acid sequence of SEQ ID NO: 47 and the other     polypeptide comprises an antibody light chain variable domain having     the light chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs:     7, 8, and 9, respectively, or an antibody light chain variable     domain having the amino acid sequence of SEQ ID NO: 47, and wherein     in the other polypeptide one or more amino acids selected from the     group consisting of K24, A25, S26, R27, N28, 129, E30, R31, Q32,     L33, A34, Q50, A51, S52, R53, K54, E55, S56, Q89, Q90, Y91, S92,     D93, P94, P95, L96, and T97, numbered according to the Kabat     numbering system, have been substituted with arbitrary amino acids     other than cysteine; -   [4] a polypeptide comprising an antibody heavy chain variable     domain, wherein the polypeptide comprises an antibody heavy chain     variable domain having the heavy chain CDR 1, 2, and 3 amino acid     sequences of SEQ ID NOs: 1, 2, and 3, respectively, or an antibody     heavy chain variable domain having the amino acid sequence of SEQ ID     NO: 45, in which one or more amino acids selected from the group     consisting of Y31, Y32, D33, 134, Q35, S50, 151, S52, P52a, S53,     G54, Q55, S56, T57, Y58, Y59, R60, R61, E62, V63, K64, G65, R95,     T96, G97, R98, E99, Y100, G100a, G100b, G100c, W100d, Y100e, F100f,     D101, and Y102, numbered according to the Kabat numbering system,     have been substituted with arbitrary amino acids other than     cysteine; -   [5] a bispecific antibody which recognizes FIX and/or FIXa, and FX,     wherein the antibody comprises an antibody heavy chain variable     domain having the heavy chain CDR 1, 2, and 3 amino acid sequences     of SEQ ID NOs: 1, 2, and 3, respectively, or comprises an antibody     heavy chain variable domain having the amino acid sequence of SEQ ID     NO: 45, in which one or more amino acids selected from the group     consisting of Y31, Y32, D33, 134, Q35, S50, 151, S52, P52a, S53,     G54, Q55, S56, T57, Y58, Y59, R60, R61, E62, V63, K64, G65, R95,     T96, G97, R98, E99, Y100, G100a, G100b, G100c, W100d, Y100e, F100f,     D101, and Y102, numbered according to the Kabat numbering system,     have been substituted with arbitrary amino acids other than     cysteine; -   [6] a polypeptide comprising an antibody heavy chain variable     domain, wherein the polypeptide comprises an antibody heavy chain     variable domain having the heavy chain CDR 1, 2, and 3 amino acid     sequences of SEQ ID NOs: 4, 5, and 6, respectively, or an antibody     heavy chain variable domain having the amino acid sequence of SEQ ID     NO: 46, in which one or more amino acids selected from the group     consisting of D31, N32, N33, M34, D35, D50, 151, N52, T52a, R53,     S54, G55, G56, S57, 158, Y59, N60, E61, E62, F63, Q64, D65, R95,     K96, S97, Y98, G99, Y100, Y100a, L100b, D101, and E102, numbered     according to the Kabat numbering system, have been substituted with     arbitrary amino acids other than cysteine; -   [7] a bispecific antibody which recognizes FIX and/or FIXa, and FX,     wherein the antibody comprises an antibody heavy chain variable     domain having the heavy chain CDR 1, 2, and 3 amino acid sequences     of SEQ ID NOs: 4, 5, and 6, respectively, or comprises an antibody     heavy chain variable domain having the amino acid sequence of SEQ ID     NO: 46, in which one or more amino acids selected from the group     consisting of D31, N32, N33, M34, D35, D50, 151, N52, T52a, R53,     S54, G55, G56, S57, 158, Y59, N60, E61, E62, F63, Q64, D65, R95,     K96, S97, Y98, G99, Y100, Y100a, L100b, D101, and E102, numbered     according to the Kabat numbering system, have been substituted with     an arbitrary amino acids other than cysteine; -   [8] a bispecific antibody which recognizes FIX and/or FIXa, and FX,     wherein a first polypeptide and a third polypeptide form a pair and     a second polypeptide and a fourth polypeptide form a pair, wherein     the second polypeptide comprises the antibody heavy chain variable     domain amino acid sequence of SEQ ID NO: 46, the third polypeptide     comprises the antibody light chain amino acid sequence of SEQ ID NO:     42, and the fourth polypeptide comprises the antibody light chain     amino acid sequence of SEQ ID NO: 44, and the first polypeptide     comprises an antibody heavy chain variable domain having the heavy     chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 1, 2, and     3, respectively, or an antibody heavy chain variable domain having     the amino acid sequence of SEQ ID NO: 45, and wherein in the first     polypeptide one or more amino acids selected from the group     consisting of Y31, Y32, D33, 134, Q35, S50, 151, S52, P52a, S53,     G54, Q55, S56, T57, Y58, Y59, R60, R61, E62, V63, K64, G65, R95,     T96, G97, R98, E99, Y100, G100a, G100b, G100c, W100d, Y100e, F100f,     D101, and Y102, numbered according to the Kabat numbering system,     have been substituted with arbitrary amino acids other than     cysteine; -   [9] a bispecific antibody which recognizes FIX and/or FIXa, and FX,     wherein a first polypeptide and a third polypeptide form a pair and     a second polypeptide and a fourth polypeptide form a pair, wherein     the first polypeptide comprises the antibody heavy chain amino acid     sequence of SEQ ID NO: 45, the third polypeptide comprises the     antibody light chain amino acid sequence of SEQ ID NO: 43, and the     fourth polypeptide comprises the antibody light chain amino acid     sequence of SEQ ID NO: 44, and the second polypeptide comprises an     antibody heavy chain variable domain having the heavy chain CDR 1,     2, and 3 amino acid sequences of SEQ ID NOs: 4, 5, and 6,     respectively, or an antibody heavy chain variable domain having the     amino acid sequence of SEQ ID NO: 46, and wherein in the second     polypeptide one or more amino acids selected from the group     consisting of D31, N32, N33, M34, D35, D50, 151, T52a, N52, R53,     S54, G55, G56, S57, 158, Y59, N60, E61, E62, F63, Q64, D65, R95,     K96, S97, Y98, G99, Y100, Y100a, L100b, D101, and E102, numbered     according to the Kabat numbering system, have been substituted with     arbitrary amino acids other than cysteine; -   [10] an antibody light chain, which comprises any one of the amino     acid sequences selected from the following (a1) to (a6), (b1) to     (b23), and (cl) to (c3):

(a1) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 13 (QNK131);

(a2) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 14 (QNK284);

(a3) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 15 (QNK315);

(a4) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 16 (QNL182);

(a5) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 17 (QNL492);

(a6) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 18 (QNL576);

(b1) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 19 (JNK131);

(b2) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 20 (JNK163);

(b3) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 21 (JNK252);

(b4) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 22 (JNK263);

(b5) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 23 (JNK339);

(b6) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 24 (JNK348);

(b7) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 25 (JNK351);

(b8) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 26 (JNK360);

(b9) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 27 (JNK378);

(b10) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 28 (JNK3 82);

(b11) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 29 (JNL03 6);

(b12) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 30 (JNL072);

(b13) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 31 (JNL095);

(b14) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 32 (JNL176);

(b15) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 33 (JNL208);

(b16) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 34 (JNL224);

(b17) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 35 (JNL260);

(b18) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 36 (JNL056);

(b19) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 37 (JNL059);

(b20) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 38 (JNL226);

(b21) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 39 (JNL250);

(b22) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 40 (JNL263);

(b23) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 41 (JNL281);

(c1) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 42 (QAL187);

(c2) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 43 (QAL201); and

(c3) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 44 (JYL280);

-   [11] a bispecific antibody which recognizes FIX and/or FIXa, and FX,     wherein a first polypeptide (d) and a third polypeptide (0 form a     pair, a second polypeptide (e) and a fourth polypeptide (g) form a     pair, and wherein each of the polypeptides is the following     polypeptide:

(d) the first polypeptide is (d1) or (d2):

-   -   (d1) a polypeptide which comprises an antibody heavy chain         variable domain having the heavy chain CDR 1, 2, and 3 amino         acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, or an         antibody heavy chain variable domain having the amino acid         sequence of SEQ ID NO: 45;     -   (d2) a polypeptide which comprises an antibody heavy chain         variable domain having the heavy chain CDR 1, 2, and 3 amino         acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, or an         antibody heavy chain variable domain having the amino acid         sequence of SEQ ID NO: 45, in which one or more amino acids         selected from the group consisting of Y31, Y32, D33, 134, Q35,         S50, 151, S52, P52a, S53, G54, Q55, S56, T57, Y58, Y59, R60,         R61, E62, V63, K64, G65, R95, T96, G97, R98, E99, Y100, G100a,         G100b, G100c, W100d, Y100e, F100f, D101, and Y102, numbered         according to the Kabat numbering system, have been substituted         with arbitrary amino acids other than cysteine;

(e) the second polypeptide is (e1) or (e2):

-   -   (e1) a polypeptide which comprises an antibody heavy chain         variable domain having the heavy chain CDR 1, 2, and 3 amino         acid sequences of SEQ ID NOs: 4, 5, and 6, respectively, or an         antibody heavy chain variable domain having the amino acid         sequence of SEQ ID NO: 46;     -   (e2) a polypeptide which comprises an antibody heavy chain         variable domain having the heavy chain CDR 1, 2, and 3 amino         acid sequences of SEQ ID NOs: 4, 5, and 6, respectively, or an         antibody heavy chain variable domain having the amino acid         sequence of SEQ ID NO: 46, in which one or more amino acids         selected from the group consisting of D31, N32, N33, M34, D35,         D50, 151, T52a, N52, R53, S54, G55, G56, S57, 158, Y59, N60,         E61, E62, F63, Q64, D65, R95, K96, S97, Y98, G99, Y100, Y100a,         L100b, D101, and E102, numbered according to the Kabat numbering         system, have been substituted with arbitrary amino acids other         than cysteine;

(f) the third polypeptide is (f1), (f2), or (f3):

-   -   (f1) a polypeptide which comprises an antibody light chain         variable domain having the light chain CDR 1, 2, and 3 amino         acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, or an         antibody light chain variable domain having the amino acid         sequence of SEQ ID NO: 47;     -   (f2) a polypeptide which comprises an antibody light chain         variable domain having the light chain CDR 1, 2, and 3 amino         acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, or an         antibody light chain variable domain having the amino acid         sequence of SEQ ID NO: 47, in which one or more amino acids         selected from the group consisting of K24, A25, S26, R27, N28,         129, E30, R31, Q32, L33, A34, Q50, A51, S52, R53, K54, E55, S56,         Q89, Q90, Y91, S92, D93, P94, P95, L96, and T97, numbered         according to the Kabat numbering system, have been substituted         with arbitrary amino acids other than cysteine;     -   (f3) the polypeptide of any one of (a1) to (a6) and (cl) to (c2)         described in [10]; and

(g) the fourth polypeptide is (g1), (g2), or (g3):

-   -   (g1) a polypeptide which comprises an antibody light chain         variable domain having the light chain CDR 1, 2, and 3 amino         acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, or an         antibody light chain variable domain having the amino acid         sequence of SEQ ID NO: 47;     -   (g2) a polypeptide which comprises an antibody light chain         variable domain having the light chain CDR 1, 2, and 3 amino         acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, or an         antibody light chain variable domain having the amino acid         sequence of SEQ ID NO: 47, in which one or more amino acids         selected from the group consisting of K24, A25, S26, R27, N28,         129, E30, R31, Q32, L33, A34, Q50, A51, S52, R53, K54, E55, S56,         Q89, Q90, Y91, S92, D93, P94, P95, L96, and T97, numbered         according to the Kabat numbering system, have been substituted         with arbitrary amino acids other than cysteine;     -   (g3) the polypeptide of any one of (b1) to (b23), and (c3)         described in [10];

-   [12] a method of producing a variant of Emicizumab, which comprises     the following step (a):

(a) performing one or more of the following substitutions (i) to (iii), wherein the numbering is according to the Kabat numbering system:

-   -   (i) substitution of one or more amino acids selected from the         group consisting of K24, A25, S26, R27, N28, 129, E30, R31, Q32,         L33, A34, Q50, A51, S52, R53, K54, E55, S56, Q89, Q90, Y91, S92,         D93, P94, P95, L96, and T97, in an antibody light chain variable         domain comprising the light chain CDR 1, 2, and 3 amino acid         sequences of SEQ ID NOs: 7, 8, and 9, respectively;     -   (ii) substitution of one or more amino acids selected from the         group consisting of Y31, Y32, D33, 134, Q35, S50, 151, S52,         P52a, S53, G54, Q55, S56, T57, Y58, Y59, R60, R61, E62, V63,         K64, G65, R95, T96, G97, R98, E99, Y100, G100a, G100b, G100c,         W100d, Y100e, F100f, D101, and Y102, in an antibody heavy chain         variable domain comprising the heavy chain CDR 1, 2, and 3 amino         acid sequences of SEQ ID NOs: 1, 2, and 3, respectively; and     -   (iii) substitution of one or more amino acids selected from the         group consisting of D31, N32, N33, M34, D35, D50, 151, T52a,         N52, R53, S54, G55, G56, S57, 158, Y59, N60, E61, E62, F63, Q64,         D65, R95, K96, S97, Y98, G99, Y100, Y100a, L100b, D101, and         E102, in an antibody heavy chain variable domain comprising the         heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs:         4, 5, and 6, respectively;

-   [13] a method of isolating a variant of Emicizumab, which comprises     the steps of (a) to (c) below:

(a) producing a variant of Emicizumab by performing one or more of the following (i) to (iii), wherein the numbering is according to the Kabat numbering system:

-   -   (i) substitution of one or more amino acids selected from the         group consisting of K24, A25, S26, R27, N28, 129, E30, R31, Q32,         L33, A34, Q50, A51, S52, R53, K54, E55, S56, Q89, Q90, Y91, S92,         D93, P94, P95, L96, and T97, in an antibody light chain variable         domain comprising the light chain CDR 1, 2, and 3 amino acid         sequences of SEQ ID NOs: 7, 8, and 9, respectively;     -   (ii) substitution of one or more amino acids selected from the         group consisting of Y31, Y32, D33, 134, Q35, S50, 151, S52,         P52a, S53, G54, Q55, S56, T57, Y58, Y59, R60, R61, E62, V63,         K64, G65, R95, T96, G97, R98, E99, Y100, G100a, G100b, G100c,         W100d, Y100e, F100f, D101, and Y102, in an antibody heavy chain         variable domain comprising the heavy chain CDR 1, 2, and 3 amino         acid sequences of SEQ ID NOs: 1, 2, and 3, respectively; and     -   (iii) substitution of one or more amino acids selected from the         group consisting of D31, N32, N33, M34, D35, D50, 151, T52a,         N52, R53, S54, G55, G56, S57, 158, Y59, N60, E61, E62, F63, Q64,         D65, R95, K96, S97, Y98, G99, Y100, Y100a, L100b, D101, and         E102, in an antibody heavy chain variable domain comprising the         heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs:         4, 5, and 6, respectively;

(b) measuring FVIII cofactor function-substituting activity of the variant produced in (a); and

(c) selecting a variant of Emicizumab having improved FVIII cofactor function-substituting activity compared to that of Emicizumab.

Furthermore, the present invention provides the following [14] to [26]:

-   [14] the polypeptide of any one of [1], [4], and [6], which is an     antibody; -   [15] the antibody of any one of [2], [3], [5], [7] to [9], [11], and     [14], which is a humanized antibody or a human antibody; -   [16] the antibody of any one of [2], [3], [5], [7] to [9], [11],     [14], and [15], which is an antibody selected from the group     consisting of Fv, Fab, Fab′, Fab′-SH, F(ab′)2, diabody, linear     antibody, single-chain antibody molecule, and multispecific antibody     formed from antibody fragments; -   [17] a nucleic acid encoding the antibody of any one of [2], [3],     [5], [7] to [9], [11], and [14] to [16]; -   [18] a vector into which the nucleic acid of [17] has been inserted; -   [19] a cell, which comprises the nucleic acid of [17] or the vector     of [18]; -   [20] a pharmaceutical formulation, which comprises the antibody of     any one of [2], [3], [5], [7] to [9], [11], and [14] to [16], and a     pharmaceutically acceptable carrier; -   [21] the pharmaceutical formulation of [20], which is used for     prevention and/or treatment of bleeding, a disease accompanying     bleeding, or a disease caused by bleeding, wherein the disease is a     disease that develops and/or progresses due to a decrease or     deficiency in the activity of blood coagulation factor VIII and/or     activated blood coagulation factor VIII; -   [22] the pharmaceutical formulation of [21], wherein the disease     that develops and/or progresses due to a decrease or deficiency in     the activity of blood coagulation factor VIII and/or activated blood     coagulation factor VIII is hemophilia A; -   [23] the pharmaceutical formulation of [22], wherein the disease     that develops and/or progresses due to a decrease or deficiency in     the activity of blood coagulation factor VIII and/or activated blood     coagulation factor VIII is a disease showing emergence of an     inhibitor against blood coagulation factor VIII and/or activated     blood coagulation factor VIII; -   [24] the pharmaceutical formulation of [21], wherein the disease     that develops and/or progresses due to a decrease or deficiency in     the activity of blood coagulation factor VIII and/or activated blood     coagulation factor VIII is acquired hemophilia; -   [25] the pharmaceutical formulation of [21], wherein the disease     that develops and/or progresses due to a decrease or deficiency in     the activity of blood coagulation factor VIII and/or activated blood     coagulation factor VIII is von Willebrand disease; and -   [26] a commercial package of a therapeutic product for use in the     method for preventing and/or treating bleeding, a disease     accompanying bleeding, or a disease caused by bleeding, which     comprises at least the antibody of any one of [2], [3], [5], [7] to     [9], [11], and [14] to [16].

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph presenting the FVIII cofactor function-substituting activities of bispecific antibodies comprising novel L chains.

FIG. 2 shows a graph presenting the FVIII cofactor function-substituting activities of Q499/QAL201//J327/JYL280 and Q499/L404//J327/L404.

MODE FOR CARRYING OUT THE INVENTION

The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

An “effective amount” of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (residues 446-447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

“Framework” or “FR” refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.

A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

The term “hypervariable region” or “HVR” as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence (“complementarity determining regions” or “CDRs”) and/or form structurally defined loops (“hypervariable loops”) and/or contain the antigen-contacting residues (“antigen contacts”). Generally, antibodies comprise six HVRs: three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). Exemplary HVRs herein include:

-   (a) hypervariable loops occurring at amino acid residues 26-32 (L1),     50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3)     (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); -   (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2),     89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al.,     Sequences of Proteins of Immunological Interest, 5th Ed. Public     Health Service, National Institutes of Health, Bethesda, Md.     (1991)); -   (c) antigen contacts occurring at amino acid residues 27c-36 (L1),     46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3)     (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and -   (d) combinations of (a), (b), and/or (c), including HVR amino acid     residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1),     26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

An “isolated” antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies composing the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

“Native antibodies” refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

The terms “FIX”, “FIXa”, and “FX” as used herein, refer to any native FIX, FIXa, and FX from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length” unprocessed FIX, FIXa, and FX as well as any form of FIX, FIXa, and FX that results from processing in the cell. The term also encompasses naturally occurring variants of FIX, FIXa, and FX, e.g., splice variants or allelic variants.

As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.

The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

Emicizumab (ACE910) which is a bispecific antibody that substitutes for the function of FVIII, and binds to both FIX and/or FIXa, and FX is described below. A bispecific antibody (Q499-z121/J327-z119/L404-k) in which a first polypeptide and a third polypeptide form a pair, and a second polypeptide and a fourth polypeptide form a pair, and in which the first polypeptide comprises an H chain comprising the H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 1, 2, and 3 (H chain CDRs of Q499), respectively, the second polypeptide comprises an H chain comprising the H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 4, 5, and 6 (H chain CDRs of J327), respectively, and the third polypeptide and the fourth polypeptide comprise a commonly shared L chain comprising the L chain CDR 1, 2, 3 amino acid sequences of SEQ ID NOs: 7, 8, and 9 (L chain CDRs of L404), respectively.

More specifically, the above-described bispecific antibody is a bispecific antibody (Q499-z121/J327-z119/L404-k) in which a first polypeptide and a third polypeptide form a pair, and a second polypeptide and a fourth polypeptide form a pair, and in which the first polypeptide comprises an H chain comprising the antibody heavy chain variable domain amino acid sequence of SEQ ID NO: 45, the second polypeptide comprises an H chain comprising the antibody heavy chain variable domain amino acid sequence of SEQ ID NO: 46, and the third polypeptide and fourth polypeptide comprise a commonly shared L chain comprising the antibody light chain variable domain amino acid sequence of SEQ ID NO: 47.

More specifically, the above-described bispecific antibody is a bispecific antibody (Q499-z121/J327-z119/L404-k) in which a first polypeptide and a third polypeptide form a pair, and a second polypeptide and a fourth polypeptide form a pair, and in which the first polypeptide comprises an H chain comprising the amino acid sequence of SEQ ID NO: 10, the second polypeptide comprises an H chain comprising the amino acid sequence of SEQ ID NO: 11, and the third polypeptide and fourth polypeptide comprise a commonly shared L chain comprising the amino acid sequence of SEQ ID NO: 12.

Such antibody can be obtained, for example, by following the methods described in WO2005/035756, WO2006/109592, WO2012/067176, and such. Herein below, Emicizumab is synonymous with ACE910 in the present specification.

In one aspect, “a variant of Emicizumab” according to the present invention refers to a polypeptide or an antibody in which at least one or more amino acid sequences of the heavy chain variable regions or light chain variable regions of Emicizumab differ from the amino acid sequences of Emicizumab, and includes any variants to the extent of being able to achieve the objective of the present invention. In one embodiment, even if all of the variable regions of the heavy chains or the light chains which are a part of a variant are different from the variable regions of Emicizumab, such variant may be included in the variants of the present invention.

For example, variants of Emicizumab are the following polypeptides and antibodies, but are not limited thereto.

In one aspect, the present invention provides a polypeptide comprising an antibody light chain variable domain, the polypeptide comprising an antibody light chain variable domain having the light chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, or an antibody light chain variable domain having the amino acid sequence of SEQ ID NO: 47, in which one or more amino acids selected from the group consisting of K24, A25, S26, R27, N28, 129, E30, R31, Q32, L33, A34, Q50, A51, S52, R53, K54, E55, S56, Q89, Q90, Y91, S92, D93, P94, P95, L96, and T97, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine.

In another aspect, the present invention provides a bispecific antibody which recognizes FIX and/or FIXa, and FX, the antibody comprising an antibody light chain variable domain having the light chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, or an antibody light chain variable domain having the amino acid sequence of SEQ ID NO: 47, in which one or more amino acids selected from the group consisting of K24, A25, S26, R27, N28, I29, E30, R31, Q32, L33, A34, Q50, A51, S52, R53, K54, E55, S56, Q89, Q90, Y91, S92, D93, P94, P95, L96, and T97, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine.

In another aspect, the present invention provides a bispecific antibody which recognizes FIX and/or FIXa, and FX, in which a first polypeptide and a third polypeptide form a pair and a second polypeptide and a fourth polypeptide form a pair, and in which the first polypeptide comprises the antibody heavy chain variable domain amino acid sequence of SEQ ID NO: 45, the second polypeptide comprises the antibody heavy chain variable domain amino acid sequence of SEQ ID NO: 46, either the third polypeptide or the fourth polypeptide comprises the antibody light chain variable domain amino acid sequence of SEQ ID NO: 47, and the other polypeptide comprises an antibody light chain variable domain having the light chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, or an antibody light chain variable domain having the amino acid sequence of SEQ ID NO: 47, and in the other polypeptide one or more amino acids selected from the group consisting of K24, A25, S26, R27, N28, 129, E30, R31, Q32, L33, A34, Q50, A51, S52, R53, K54, E55, S56, Q89, Q90, Y91, S92, D93, P94, P95, L96, and T97, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine.

In another aspect, the present invention provides a polypeptide comprising an antibody heavy chain variable domain, the polypeptide comprising an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, or an antibody heavy chain variable domain having the amino acid sequence of SEQ ID NO: 45, in which one or more amino acids selected from the group consisting of Y31, Y32, D33, I34, Q35, S50, 151, S52, P52a, S53, G54, Q55, S56, T57, Y58, Y59, R60, R61, E62, V63, K64, G65, R95, T96, G97, R98, E99, Y100, G100a, G100b, G100c, W100d, Y100e, F100f, D101, and Y102, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine.

In another aspect, the present invention provides a bispecific antibody which recognizes FIX and/or FIXa, and FX, the antibody comprising an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, or an antibody heavy chain variable domain having the amino acid sequence of SEQ ID NO: 45, in which one or more amino acids selected from the group consisting of Y31, Y32, D33, 134, Q35, S50, I51, S52, P52a, S53, G54, Q55, S56, T57, Y58, Y59, R60, R61, E62, V63, K64, G65, R95, T96, G97, R98, E99, Y100, G100a, G100b, G100c, W100d, Y100e, F100f, D101, and Y102, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine.

In another aspect, the present invention provides a polypeptide comprising an antibody heavy chain variable domain, the polypeptide comprising an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively, or an antibody heavy chain variable domain having the amino acid sequence of SEQ ID NO: 46, in which one or more amino acids selected from the group consisting of D31, N32, N33, M34, D35, D50, 151, N52, T52a, R53, S54, G55, G56, S57, 158, Y59, N60, E61, E62, F63, Q64, D65, R95, K96, S97, Y98, G99, Y100, Y100a, L100b, D101, and E102, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine.

In another aspect, the present invention provides a bispecific antibody which recognizes FIX and/or FIXa, and FX, the antibody comprising an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively, or an antibody heavy chain variable domain having the amino acid sequence of SEQ ID NO: 46, in which one or more amino acids selected from the group consisting of D31, N32, N33, M34, D35, D50, 151, N52, T52a, R53, S54, G55, G56, S57, I58, Y59, N60, E61, E62, F63, Q64, D65, R95, K96, S97, Y98, G99, Y100, Y100a, L100b, D101, and E102, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine.

In another aspect, the present invention provides a bispecific antibody which recognizes FIX and/or FIXa, and FX, in which a first polypeptide and a third polypeptide form a pair and a second polypeptide and a fourth polypeptide form a pair, and in which the second polypeptide comprises the antibody heavy chain variable domain amino acid sequence of SEQ ID NO: 46, the third polypeptide comprises the antibody light chain amino acid sequence of SEQ ID NO: 42, the fourth polypeptide comprises the antibody light chain amino acid sequence of SEQ ID NO: 44, and the first polypeptide comprises an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acids of SEQ ID NOs: 1, 2, and 3, respectively, or an antibody heavy chain variable domain having the amino acid sequence of SEQ ID NO: 45, and in the first polypeptide one or more amino acids selected from the group consisting of Y31, Y32, D33, 134, Q35, S50, I51, S52, P52a, S53, G54, Q55, S56, T57, Y58, Y59, R60, R61, E62, V63, K64, G65, R95, T96, G97, R98, E99, Y100, G100a, G100b, G100c, W100d, Y100e, F100f, D101, and Y102, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine.

In another aspect, the present invention provides a bispecific antibody which recognizes FIX and/or FIXa, and FX, in which a first polypeptide and a third polypeptide form a pair and a second polypeptide and a fourth polypeptide form a pair, and in which the first polypeptide comprises the antibody heavy chain amino acid sequence of SEQ ID NO: 45, the third polypeptide comprises the antibody light chain amino acid sequence of SEQ ID NO: 43, the fourth polypeptide comprises the antibody light chain amino acid sequence of SEQ ID NO: 44, and the second polypeptide comprises an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively, or an antibody heavy chain variable domain having the amino acid sequence of SEQ ID NO: 46, and in the second polypeptide one or more amino acids selected from the group consisting of D31, N32, N33, M34, D35, D50, I51, T52a, N52, R53, S54, G55, G56, S57, 158, Y59, N60, E61, E62, F63, Q64, D65, R95, K96, S97, Y98, G99, Y100, Y100a, L100b, D101, and E102, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine.

In another aspect, the present invention provides an antibody light chain which is a novel antibody light chain comprising any one of the amino acid sequences selected from the following (a1) to (a6) and (b1) to (b23). The light chains can be used as a light chain that substitutes for the commonly shared light chain included in Emicizumab. The following light chains of (a1) to (a6) are examples of light chains that bind to FIX and/or FIXa, and the following L chains of (b1) to (b23) are examples of light chains that bind to FX:

-   (a1) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 13 (QNK131); -   (a2) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 14 (QNK284); -   (a3) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 15 (QNK315); -   (a4) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 16 (QNL182); -   (a5) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 17 (QNL492); -   (a6) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 18 (QNL576); -   (b1) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 19 (JNK131); -   (b2) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 20 (JNK163); -   (b3) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 21 (JNK252); -   (b4) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 22 (JNK263); -   (b5) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 23 (JNK339); -   (b6) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 24 (JNK348); -   (b7) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 25 (JNK351); -   (b8) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 26 (JNK360); -   (b9) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 27 (JNK378); -   (b10) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 28 (JNK3 82); -   (b11) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 29 (JNL036); -   (b12) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 30 (JNL072); -   (b13) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 31 (JNL095); -   (b14) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 32 (JNL176); -   (b15) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 33 (JNL208); -   (b16) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 34 (JNL224); -   (b17) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 35 (JNL260); -   (b18) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 36 (JNL056); -   (b19) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 37 (JNL059); -   (b20) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 38 (JNL226); -   (b21) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 39 (JNL250); -   (b22) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 40 (JNL263); and -   (b23) an antibody light chain comprising the amino acid sequence of     SEQ ID NO: 41 (JNL281).

In specific embodiments, the present invention provides variants of the above-mentioned antibody L chains of (a1) to (a6) and (b1) to (b23).

In one embodiment, the present invention provides the following antibody L chain variants:

-   (c1) an antibody light chain (QAL187) having the amino acid sequence     of SEQ ID NO: 42, which is a variant of the antibody light chain of     SEQ ID NO: 13; -   (c2) an antibody L chain (QAL201) having the amino acid sequence of     SEQ ID NO: 43, which is a variant of the antibody light chain of SEQ     ID NO: 13; and -   (c3) an antibody L chain (JYL280) having the amino acid sequence of     SEQ ID NO: 44, which is a variant of the antibody light chain of SEQ     ID NO: 31.

In another aspect, the present invention provides a bispecific antibody which recognizes FIX and/or FIXa, and FX, in which a first polypeptide (d) and a third polypeptide (0 form a pair, a second polypeptide (e) and a fourth polypeptide (g) form a pair, and the respective polypeptides are the following polypeptides:

(d) the first polypeptide is (d1) or (d2):

-   -   (d1) a polypeptide which comprises an antibody heavy chain         variable domain having the heavy chain CDR 1, 2, and 3 amino         acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, or an         antibody heavy chain variable domain having the amino acid         sequence of SEQ ID NO: 45; or

-   (d2) a polypeptide which comprises an antibody heavy chain variable     domain having the heavy chain CDR 1, 2, and 3 amino acid sequences     of SEQ ID NOs: 1, 2, and 3, respectively, or an antibody heavy chain     variable domain having the amino acid sequence of SEQ ID NO: 45, in     which one or more amino acids selected from the group consisting of     Y31, Y32, D33, I34, Q35, S50, 151, S52, P52a, S53, G54, Q55, S56,     T57, Y58, Y59, R60, R61, E62, V63, K64, G65, R95, T96, G97, R98,     E99, Y100, G100a, G100b, G100c, W100d, Y100e, F100f, D101, and Y102,     numbered according to the Kabat numbering system, have been     substituted with arbitrary amino acids other than cysteine;

(e) the second polypeptide is (e1) or (e2):

-   -   (e1) a polypeptide which comprises an antibody heavy chain         variable domain having the heavy chain CDR 1, 2, and 3 amino         acid sequences of SEQ ID NOs: 4, 5, and 6, respectively, or an         antibody heavy chain variable domain having the amino acid         sequence of SEQ ID NO: 46; or     -   (e2) a polypeptide which comprises an antibody heavy chain         variable domain having the heavy chain CDR 1, 2, and 3 amino         acid sequences of SEQ ID NOs: 4, 5, and 6, respectively, or an         antibody heavy chain variable domain having the amino acid         sequence of SEQ ID NO: 46, in which one or more amino acids         selected from the group consisting of D31, N32, N33, M34, D35,         D50, I51, T52a, N52, R53, S54, G55, G56, S57, 158, Y59, N60,         E61, E62, F63, Q64, D65, R95, K96, S97, Y98, G99, Y100, Y100a,         L100b, D101, and E102, numbered according to the Kabat numbering         system, have been substituted with arbitrary amino acids other         than cysteine;

(f) the third polypeptide is (f1), (f2), or (f3):

-   -   (f1) a polypeptide which comprises an antibody light chain         variable domain having the light chain CDR 1, 2, and 3 amino         acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, or an         antibody light chain variable domain having the amino acid         sequence of SEQ ID NO: 47;     -   (f2) a polypeptide which comprises an antibody light chain         variable domain having the light chain CDR 1, 2, and 3 amino         acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, or an         antibody light chain variable domain having the amino acid         sequence of SEQ ID NO: 47, in which one or more amino acids         selected from the group consisting of K24, A25, S26, R27, N28,         I29, E30, R31, Q32, L33, A34, Q50, A51, S52, R53, K54, E55, S56,         Q89, Q90, Y91, S92, D93, P94, P95, L96, and T97, numbered         according to the Kabat numbering system, have been substituted         with arbitrary amino acids other than cysteine; or     -   (f3) the polypeptide of any one of (a1) to (a6) and (c1) to (c2)         described herein; and

(g) the fourth polypeptide is (g1), (g2), or (g3):

-   -   (g1) a polypeptide which comprises an antibody light chain         variable domain having the light chain CDR 1, 2, and 3 amino         acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, or an         antibody light chain variable domain having the amino acid         sequence of SEQ ID NO: 47;     -   (g2) a polypeptide which comprises an antibody light chain         variable domain having the light chain CDR 1, 2, and 3 amino         acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, or an         antibody light chain variable domain having the amino acid         sequence of SEQ ID NO: 47, in which one or more amino acids         selected from the group consisting of K24, A25, S26, R27, N28,         I29, E30, R31, Q32, L33, A34, Q50, A51, S52, R53, K54, E55, S56,         Q89, Q90, Y91, S92, D93, P94, P95, L96, and T97, numbered         according to the Kabat numbering system, have been substituted         with arbitrary amino acids other than cysteine; or     -   (g3) the polypeptide of any one of (b1) to (b23), and (c3)         described herein.

In one aspect, the present invention provides a polypeptide or an antibody, in which at least one or more amino acid residues at the following positions in an antibody light chain variable domain having the light chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, or at least one or more amino acid residues at the following positions in the antibody light chain variable domain of SEQ ID NO: 47 are any of the following amino acid residues.

In the above-mentioned polypeptide or antibody, the number of amino acid residues that are different from an antibody light chain variable domain having the light chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, or the number of amino acid residues that are different from the antibody light chain variable domain of SEQ ID NO: 47 is one or more, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and more preferably 21 or more. The position of substitution is indicated by the position numbered according to the Kabat numbering system.

-   K24: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, R, F, W, Y -   A25: I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, Y -   S26: A, I, L, M, P, V, G, N, Q, T, D, E, H, K, R, F, W, Y -   R27: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, F, W, Y -   N28: A, I, L, M, P, V, G, Q, S, T, D, E, H, K, R, F, W, Y -   I29: A, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, Y -   E30: A, I, L, M, P, V, G, N, Q, S, T, D, H, K, R, F, W, Y -   R31: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, F, W, Y -   Q32: A, I, L, M, P, V, G, N, S, T, D, E, H, K, R, F, W, Y -   L33: A, I, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, Y -   A34: I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, Y -   Q50: A, I, L, M, P, V, G, N, S, T, D, E, H, K, R, F, W, Y -   A51: I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, Y -   S52: A, I, L, M, P, V, G, N, Q, T, D, E, H, K, R, F, W, Y -   R53: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, F, W, Y -   K54: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, R, F, W, Y -   E55: A, I, L, M, P, V, G, N, Q, S, T, D, H, K, R, F, W, Y -   S56: A, I, L, M, P, V, G, N, Q, T, D, E, H, K, R, F, W, Y -   Q89: A, I, L, M, P, V, G, N, S, T, D, E, H, K, R, F, W, Y -   Q90: A, I, L, M, P, V, G, N, S, T, D, E, H, K, R, F, W, Y -   Y91: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W -   S92: A, I, L, M, P, V, G, N, Q, T, D, E, H, K, R, F, W, Y -   D93: A, I, L, M, P, V, G, N, Q, S, T, E, H, K, R, F, W, Y -   P94: A, I, L, M, V, G, N, Q, S, T, D, E, H, K, R, F, W, Y -   P95: A, I, L, M, V, G, N, Q, S, T, D, E, H, K, R, F, W, Y -   L96: A, I, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, Y -   T97: A, I, L, M, P, V, G, N, Q, S, D, E, H, K, R, F, W, Y

In one aspect, the present invention provides a polypeptide or an antibody, in which at least one or more amino acid residues at the following positions in an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, or at least one or more amino acid residues at the following positions in the antibody heavy chain variable domain of SEQ ID NO: 45 are any of the following amino acid residues.

In the above-mentioned polypeptide or antibody, the number of amino acid residues that are different from an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, or the number of amino acid residues that are different from an antibody heavy chain variable domain of SEQ ID NO: 45 is one or more, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and more preferably 21 or more. The position of substitution is indicated by the position numbered according to the Kabat numbering system.

-   Y31: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W -   Y32: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W -   D33: A, I, L, M, P, V, G, N, Q, S, T, E, H, K, R, F, W, Y -   I34: A, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, Y -   Q35: A, I, L, M, P, V, G, N, S, T, D, E, H, K, R, F, W, Y -   S50: A, I, L, M, P, V, G, N, Q, T, D, E, H, K, R, F, W, Y -   I51: A, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, Y -   S52: A, I, L, M, P, V, G, N, Q, T, D, E, H, K, R, F, W, Y -   P52a: A, I, L, M, V, G, N, Q, S, T, D, E, H, K, R, F, W, Y -   S53: A, I, L, M, P, V, G, N, Q, T, D, E, H, K, R, F, W, Y -   G54: A, I, L, M, P, V, N, Q, S, T, D, E, H, K, R, F, W, Y -   Q55: A, I, L, M, P, V, G, N, S, T, D, E, H, K, R, F, W, Y -   S56: A, I, L, M, P, V, G, N, Q, T, D, E, H, K, R, F, W, Y -   T57: A, I, L, M, P, V, G, N, Q, S, D, E, H, K, R, F, W, Y -   Y58: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W -   Y59: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W -   R60: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, F, W, Y -   R61: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, F, W, Y -   E62: A, I, L, M, P, V, G, N, Q, S, T, D, H, K, R, F, W, Y -   V63: A, I, L, M, P, G, N, Q, S, T, D, E, H, K, R, F, W, Y -   K64: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, R, F, W, Y -   G65: A, I, L, M, P, V, N, Q, S, T, D, E, H, K, R, F, W, Y -   R95: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, F, W, Y -   T96: A, I, L, M, P, V, G, N, Q, S, D, E, H, K, R, F, W, Y -   G97: A, I, L, M, P, V, N, Q, S, T, D, E, H, K, R, F, W, Y -   R98: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, F, W, Y -   E99: A, I, L, M, P, V, G, N, Q, S, T, D, H, K, R, F, W, Y -   Y100: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W -   G100a: A, I, L, M, P, V, N, Q, S, T, D, E, H, K, R, F, W, Y -   G100b: A, I, L, M, P, V, N, Q, S, T, D, E, H, K, R, F, W, Y -   G100c: A, I, L, M, P, V, N, Q, S, T, D, E, H, K, R, F, W, Y -   W100d: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, Y -   Y100e: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W -   F100f: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, W, Y -   D101: A, I, L, M, P, V, G, N, Q, 5, T, E, H, K, R, F, W, Y -   Y102: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W

In one aspect, the present invention provides a polypeptide or an antibody, in which at least one or more amino acid residues at the following positions in an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively, or at least one or more amino acid residues at the following positions in the antibody heavy chain variable domain of SEQ ID NO: 46 are any of the following amino acid residues.

In the above-mentioned polypeptide or antibody, the number of amino acid residues that are different from an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively, or the number of amino acid residues that are different from an antibody heavy chain variable domain of SEQ ID NO: 46 is one or more, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and more preferably 21 or more. The position of substitution is indicated by the position numbered according to the Kabat numbering system.

-   D31: A, I, L, M, P, V, G, N, Q, S, T, E, H, K, R, F, W, Y -   N32: A, I, L, M, P, V, G, Q, S, T, D, E, H, K, R, F, W, Y -   N33: A, I, L, M, P, V, G, Q, S, T, D, E, H, K, R, F, W, Y -   M34: A, I, L, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, Y -   D35: A, I, L, M, P, V, G, N, Q, S, T, E, H, K, R, F, W, Y -   D50: A, I, L, M, P, V, G, N, Q, S, T, E, H, K, R, F, W, Y -   I51: A, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, Y -   N52: A, I, L, M, P, V, G, Q, S, T, D, E, H, K, R, F, W, Y -   T52a: A, I, L, M, P, V, G, N, Q, S, D, E, H, K, R, F, W, Y -   R53: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, F, W, Y -   S54: A, I, L, M, P, V, G, N, Q, T, D, E, H, K, R, F, W, Y -   G55: A, I, L, M, P, V, N, Q, S, T, D, E, H, K, R, F, W, Y -   G56: A, I, L, M, P, V, N, Q, S, T, D, E, H, K, R, F, W, Y -   S57: A, I, L, M, P, V, G, N, Q, T, D, E, H, K, R, F, W, Y -   I58: A, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, Y -   Y59: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W -   N60: A, I, L, M, P, V, G, Q, S, T, D, E, H, K, R, F, W, Y -   E61: A, I, L, M, P, V, G, N, Q, S, T, D, H, K, R, F, W, Y -   E62: A, I, L, M, P, V, G, N, Q, S, T, D, H, K, R, F, W, Y -   F63: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, W, Y -   Q64: A, I, L, M, P, V, G, N, S, T, D, E, H, K, R, F, W, Y -   D65: A, I, L, M, P, V, G, N, Q, S, T, E, H, K, R, F, W, Y -   R95: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, F, W, Y -   K96: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, R, F, W, Y -   S97: A, I, L, M, P, V, G, N, Q, T, D, E, H, K, R, F, W, Y -   Y98: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W -   G99: A, I, L, M, P, V, N, Q, S, T, D, E, H, K, R, F, W, Y -   Y100: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W -   Y100a: A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W -   L100b: A, I, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, Y -   D101: A, I, L, M, P, V, G, N, Q, 5, T, E, H, K, R, F, W, Y -   E102: A, I, L, M, P, V, G, N, Q, S, T, D, H, K, R, F, W, Y

In one embodiment, the present invention provides a bispecific antibody which recognizes FIX and/or FIXa, and FX, in which a first polypeptide and a third polypeptide form a pair and a second polypeptide and a fourth polypeptide form a pair, and in which the first polypeptide comprises the antibody heavy chain variable domain amino acid sequence of SEQ ID NO: 45, the second polypeptide comprises the antibody heavy chain variable domain amino acid sequence of SEQ ID NO: 46, the fourth polypeptide comprises the antibody light chain variable domain amino acid sequence of SEQ ID NO: 47, and the third polypeptide comprises an antibody light chain comprising any one of the amino acid sequences selected from the above-mentioned (a1) to (a6).

In another embodiment, the present invention provides a bispecific antibody which recognizes FIX and/or FIXa, and FX, in which a first polypeptide and a third polypeptide form a pair and a second polypeptide and a fourth polypeptide form a pair, and in which the first polypeptide comprises the antibody heavy chain variable domain amino acid sequence of SEQ ID NO: 45, the third polypeptide comprises the antibody light chain variable domain amino acid sequence of SEQ ID NO: 47, the second polypeptide comprises the antibody heavy chain variable domain amino acid sequence of SEQ ID NO: 46, and the fourth polypeptide comprises an antibody light chain comprising any one of the amino acid sequences selected from the above-mentioned (b1) to (b23).

In a specific embodiment, the present invention provides variants of the polypeptides and antibodies provided herein.

In specific embodiments, variants provided herein include variants that have been subjected to amino acid residue deletions in an amino acid sequence, and/or amino acid residue insertions to an amino acid sequence, and/or amino acid residue substitutions in an amino acid sequence. On the premise that the final construct will have improved FVIII cofactor function-substituting activity, arbitrary combinations of deletions, insertions, and substitutions may be performed to reach the final construct.

A. Exemplary Anti-FIX(a)/FX Bispecific Antibodies

Examples of bispecific antibodies recognizing FIX and/or FIXa, and FX (herein below simply referred to as “anti-FIX(a)/FX bispecific antibodies”) provided herein are described below; the followings are similarly applied to the other antibodies and polypeptides provided herein. Herein, FIX(a) means FIX and/or FIXa.

1. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab′, Fab′-SH, F(ab′)2, Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, N.Y.), pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(ab′)₂ fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g., E. coli or phage), as described herein.

2. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Pat. No. 5,770,429 describing HuMAB (registered trademark) technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE (registered trademark) technology, and U.S. Patent Application Publication No. US 2007/0061900, describing VELOCIMOUSE (registered trademark) technology). Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Pat. No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

3. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., 2001) and further described, e.g., in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein. Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO 93/08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using “diabody” technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991).

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading of “preferred substitutions.” More substantial changes are provided in Table 1 under the heading of “exemplary substitutions,” and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved FVIII cofactor function-substituting activity, ADCC or CDC.

TABLE 1 Original Exemplary Preferred Residue Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased FVIII cofactor function-substituting activity or affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce FVIII cofactor function-substituting activity may be made in HVRs.

A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex may be analyzed to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion of an enzyme (e.g., for ADEPT) or a polypeptide which increases the plasma half-life of the antibody to the N- or C-terminus of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located +/−3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No. US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al., especially at Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006);

and WO 2003/085107).

Antibodies variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks Fc gamma R binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express Fc gamma RIII only, whereas monocytes express Fc gamma RI, Fc gamma RH and Fc gamma RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays methods may be employed (see, for example, ACT1™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.); and CytoTox 96 (registered trademark) non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad Sci. USA 95:652-656 (1998). C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S. B. et al., Intl. Immunol. 18(12):1759-1769 (2006)).

Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).

Certain antibody variants with increased or decreased binding to FcRs are described. (See U.S. Pat. No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In some embodiments, alterations that result in altered (i.e., either increased or decreased) C1q binding and/or Complement Dependent Cytotoxicity (CDC) are made in the Fc region, e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

Antibodies with increased half lives and increased binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which increase binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, 428, 434 or 436, e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

Furthermore, Fc region variants having suppressed binding to a rheumatoid factor can be used as the antibodies of the present invention. Such variants are described in WO 2013/046704, and their examples include variants having a combination of mutations indicated by Q438R/S440E, Q438R/S440D, Q438K/S440E, or Q438K/S440D in both of their H chains.

Furthermore, Fc region variants having enhanced binding to FcRn and suppressed binding to a rheumatoid factor can be used as the antibodies of the present invention. More specifically, examples include Fc region variants having in both of their H chains, a combination of mutations shown in any one of the following a) to f).

-   a) N434A/Y436T/Q438R/S440E -   b) N434A/Y436V/Q438R/S440E -   c) M428L/N434A/Y436T/Q438R/S440E -   d) M428L/N434A/Y436V/Q438R/S440E -   e) M428L/N434A/Q438R/S440E -   f) N434A/Q438R/S440E

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351 concerning other examples of Fc region variants.

d) Antibody Derivatives

In certain embodiments, an antibody provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment, isolated nucleic acid encoding an anti-FIX(a)/FX bispecific antibody described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp2/0 cell). In one embodiment, a method of making an anti-FIX(a)/FX bispecific antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

For recombinant production of an anti-FIX(a)/FX bispecific antibody, nucleic acid encoding an antibody, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 0605672); TRI cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003).

C. Assays

Anti-FIX(a)/FX bispecific antibodies provided herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art.

1. Binding Assays and Other Assays

In one aspect, an antibody of the invention is tested for its antigen binding activity, e.g., by known methods such as ELISA, Western blot, etc.

2. Activity Assays

In one aspect, assays are provided for identifying anti-FIX(a)/FX bispecific antibodies thereof having biological activity. Biological activity may include, e.g., FVIII cofactor function-substituting activity. Antibodies having such biological activity in vivo and/or in vitro are also provided.

In certain embodiments, an antibody of the invention is tested for such biological activity.

In the present invention, “FVIII cofactor function-substituting activity”, “FVIII-substituting activity” and “activity that substitutes for FVIII function” are used synonymously, and refer to the activity of recognizing FIX and/or FIXa, and FX, and promoting FX activation (promoting FXa production).

“FVIII cofactor function-substituting activity” in the present invention means, for example, the activity calculated from FIXa-mediated FX activation reaction assay using a colorimetric determination method and thrombin generation assay using hemophilia A plasma. More specifically, the activity can be confirmed by performing evaluations using an antibody of the present invention, and, for example, a measurement system containing FIXa, FX, synthetic substrate S-2222 (synthetic substrate of FXa), and phospholipids. This measurement system shows the correlation between disease severity and clinical symptoms in cases of hemophilia A (Rosen S, Andersson M, Blomba{umlaut over ( )}ck M et al. Clinical applications of a chromogenic substrate method for determination of FVIII activity. Thromb Haemost 1985; 54: 811-23).

D. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-FIX(a)/FX bispecific antibody as described herein are prepared by mixing such antibody having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX (registered trademark), Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

E. Therapeutic Methods and Compositions

Any of the anti-FIX(a)/FX bispecific antibodies provided herein may be used in therapeutic methods.

In one aspect, an anti-FIX(a)/FX bispecific antibody for use as a medicament is provided. In further aspects, an anti-FIX(a)/FX bispecific antibody for use in treating bleeding, diseases accompanying bleeding, or diseases caused by bleeding is provided. In certain embodiments, an anti-FIX(a)/FX bispecific antibody for use in a method of treatment is provided. In certain embodiments, the invention provides an anti-FIX(a)/FX bispecific antibody for use in a method of treating an individual having bleeding, diseases accompanying bleeding, or diseases caused by bleeding comprising administering to the individual an effective amount of the anti-FIX(a)/FX bispecific antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In further embodiments, the invention provides an anti-FIX(a)/FX bispecific antibody for use in substituting for FVIII function. In certain embodiments, the invention provides an anti-FIX(a)/FX bispecific antibody for use in a method of substituting for FVIII function in an individual comprising administering to the individual an effective amount of the anti-FIX(a)/FX bispecific antibody to substitute for FVIII function. An “individual” according to any of the above embodiments is preferably a human.

In one embodiment of antibodies of the present invention, since the antibodies of the present invention have a function of substituting for cofactor FVIII, they are expected to become effective pharmaceutical agents against diseases resulting from decrease in activity (function) of this cofactor. Examples of the above-mentioned diseases include bleeding, diseases accompanying bleeding, or diseases caused by bleeding, and preferred examples are diseases that develop and/or progress due to decrease or deficiency in the activity of FVIII and/or activated blood coagulation factor VIII (FVIIIa). Such diseases include, for example, hemophilia A, diseases in which inhibitors against FVIII/FVIIIa appear, acquired hemophilia, von Willebrand disease, and such, but are not particularly limited thereto.

In a further aspect, the invention provides for the use of an anti-FIX(a)/FX bispecific antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treatment of bleeding, diseases accompanying bleeding, or diseases caused by bleeding. In a further embodiment, the medicament is for use in a method of treating bleeding, diseases accompanying bleeding, or diseases caused by bleeding comprising administering to an individual having bleeding, diseases accompanying bleeding, or diseases caused by bleeding an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In a further embodiment, the medicament is for substituting for FVIII function. In a further embodiment, the medicament is for use in a method of substituting for FVIII function in an individual comprising administering to the individual an effective amount of the medicament to substitute for FVIII function. An “individual” according to any of the above embodiments may be a human.

In a further aspect, the invention provides a method for treating a bleeding, diseases accompanying bleeding, or diseases caused by bleeding. In one embodiment, the method comprises administering to an individual having such bleeding, diseases accompanying bleeding, or diseases caused by bleeding an effective amount of an anti-FIX(a)/FX bispecific antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. An “individual” according to any of the above embodiments may be a human.

In a further aspect, the invention provides a method for substituting for FVIII function in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-FIX(a)/FX bispecific antibody to substitute for FVIII function. In one embodiment, an “individual” is a human.

In a further aspect, the invention provides pharmaceutical formulations comprising any of the anti-FIX(a)/FX bispecific antibodies provided herein, e.g., for use in any of the above therapeutic methods. In one embodiment, a pharmaceutical formulation comprises any of the anti-FIX(a)/FX bispecific antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical formulation comprises any of the anti-FIX(a)/FX bispecific antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.

Antibodies of the invention can be used either alone or in combination with other agents in a therapy. For instance, an antibody of the invention may be co-administered with at least one additional therapeutic agent. In certain embodiments, an additional therapeutic agent is a FVIII formulation.

Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the antibody of the invention can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent or agents. In one embodiment, administration of the anti-FIX(a)/FX bispecific antibody and administration of an additional therapeutic agent occur within about one month, or within about one, two or three weeks, or within about one, two, three, four, five, or six days, of each other.

An antibody of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g., by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

Antibodies of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibody needs not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of an antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 micro g/kg to 15 mg/kg (e.g., 0.1 mg/kg-10 mg/kg) of antibody can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 micro g/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the antibody would be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g., every week or every three weeks (e.g., such that the patient receives from about two to about twenty, or e.g., about six doses of the antibody). An initial higher loading dose, followed by one or more lower doses may be administered. The progress of this therapy is easily monitored by conventional techniques and assays.

F. Articles of Manufacture

In another aspect of the invention, an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label on or a package insert associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active ingredient in the composition is an antibody of the invention. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

In one aspect, the present invention is a method of producing a variant of Emicizumab, which comprises the following step (a):

(a) performing one or more of the following substitutions (i) to (iii), wherein the numbering is according to the Kabat numbering system:

(i) substitution of one or more amino acids selected from the group consisting of K24, A25, S26, R27, N28, 129, E30, R31, Q32, L33, A34, Q50, A51, S52, R53, K54, E55, S56, Q89, Q90, Y91, S92, D93, P94, P95, L96, and T97, in an antibody light chain variable domain comprising the light chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively;

(ii) substitution of one or more amino acids selected from the group consisting of Y31, Y32, D33, 134, Q35, S50, I51, S52, P52a, S53, G54, Q55, S56, T57, Y58, Y59, R60, R61, E62, V63, K64, G65, R95, T96, G97, R98, E99, Y100, G100a, G100b, G100c, W100d, Y100e, F100f, D101, and Y102, in an antibody heavy chain variable domain comprising the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively; and

(iii) substitution of one or more amino acids selected from the group consisting of D31, N32, N33, M34, D35, D50, I51, T52a, N52, R53, S54, G55, G56, S57, 158, Y59, N60, E61, E62, F63, Q64, D65, R95, K96, S97, Y98, G99, Y100, Y100a, L100b, D101, and E102, in an antibody heavy chain variable domain comprising the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

The sites of substitution described above can be one or more, or they may be a combination of (i) and (ii), a combination of (i) and (iii), a combination of (ii) and (iii), or a combination of (i), (ii), and (iii).

In one aspect, the present invention is a method of isolating a variant of Emicizumab, which comprises the following steps (a) to (c):

(a) producing a variant of Emicizumab by performing one or more of the following (i) to (iii), wherein the numbering is according to the Kabat numbering system:

(i) substitution of one or more amino acids selected from the group consisting of K24, A25, S26, R27, N28, 129, E30, R31, Q32, L33, A34, Q50, A51, S52, R53, K54, E55, S56, Q89, Q90, Y91, S92, D93, P94, P95, L96, and T97, in an antibody light chain variable domain comprising the light chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively;

(ii) substitution of one or more amino acids selected from the group consisting of Y31, Y32, D33, I34, Q35, S50, I51, S52, P52a, S53, G54, Q55, S56, T57, Y58, Y59, R60, R61, E62, V63, K64, G65, R95, T96, G97, R98, E99, Y100, G100a, G100b, G100c, W100d, Y100e, F100f, D101, and Y102, in an antibody heavy chain variable domain comprising the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively; and

(iii) substitution of one or more amino acids selected from the group consisting of D31, N32, N33, M34, D35, D50, I51, T52a, N52, R53, S54, G55, G56, S57, 158, Y59, N60, E61, E62, F63, Q64, D65, R95, K96, S97, Y98, G99, Y100, Y100a, L100b, D101, and E102, in an antibody heavy chain variable domain comprising the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively;

(b) measuring FVIII cofactor function-substituting activity of the antibody variant produced in step (a); and

(c) selecting an Emicizumab antibody variant having improved FVIII cofactor function-substituting activity compared to that of Emicizumab.

The sites of substitution described above can be one or more, or they may be a combination of (i) and (ii), a combination of (i) and (iii), a combination of (ii) and (iii), or a combination of (i), (ii), and (iii).

EXAMPLES

The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.

Example 1 Screening of Variant Antibodies for Improvement of FVIII Cofactor Function-Substituting Activity

ACE910 is a humanized IgG4 antibody showing FVIII cofactor function-substituting activity and comprising anti-FIX(a) and anti-FX domains, the antibody being composed of two types of heavy chains (Q499 and J327) which recognize FIX(a) and FX, respectively, and commonly shared L chains (L404) (heavy chain SEQ ID NOs: 45 and 46, and light chain SEQ ID NO: 47). The present inventors comprehensively introduced amino acid modifications into L404 by methods known to those skilled in the art such as PCR for introducing mutations and performed a large-scale screening of FVIII cofactor function-substituting activity, thereby discovering L-chain amino acid substitutions that improve the FVIII cofactor function-substituting activity of ACE910.

Substitution variants were produced by modifying amino acids in all of the CDRs of L404 (SEQ ID NO: 47) in the L chain of the anti-FX antibody side (heavy chain SEQ ID NO: 46 and light chain SEQ ID NO: 47) of ACE910 to amino acids other than cysteine, with fixing the L chain of the anti-FIX(a) antibody side (heavy chain SEQ ID NO: 45 and light chain SEQ ID NO: 47) of ACE910 to L404. Furthermore, substitution variants were produced by modifying amino acids in all of the CDRs of L404 in the L chain of the anti-FIX(a) antibody side to amino acids other than cysteine, with fixing the L chain of the anti-FX antibody side (heavy chain SEQ ID NO: 46 and light chain SEQ ID NO: 47) to L404 of ACE910. Expression and purification of the bispecific antibodies were carried out by methods known to those skilled in the art.

Using the various purified bispecific antibodies, their FVIII cofactor function-substituting activities were evaluated by a method known to those skilled in the art. Specifically, measurements were performed by the following method. All reactions were carried out at room temperature. Five μL of antibody solution diluted with Tris-buffered saline containing 0.1% bovine serum albumin (hereafter referred to as TBSB) was mixed with 5 μL of 600 ng/mL Human Factor IXa beta (Enzyme Research Laboratories) and then incubated in a 384-well plate at room temperature for 30 minutes. The enzyme reaction in this mixture was initiated by adding 5 μL of 24.7 μg/mL Human Factor X (Enzyme Research Laboratories), and four minutes later, 5 μL of 0.5 M EDTA was added to stop the reaction. The coloring reaction was initiated by adding 5 μL of coloring substrate solution (S-2222, SEKISUI MEDICAL). After a 30-minute coloring reaction, the change in absorbance at 405 nm was measured using the SpectraMax 340PC384 (Molecular Devices).

As a result, several amino acid substitutions indicated in Table 2 below were found to be able to improve the FVIII cofactor function-substituting activity as compared with the parent antibody ACE910. Table 2 shows the mutated positions indicated by Kabat numbering (the number in the “position” column on the far left), the amino acids in ACE910 (amino acids (one letter code) in the second column from the left), amino acids after the mutation (amino acids (one letter code) in the uppermost row), and values (each of the numerical values in the table) obtained by calculating the specific activity relative to ACE910 (purified antibody) and applying a correction using the value of specific activity of ACE910 (culture supernatant). In Table 2, (−) indicates that the expression level of the antibody was low, and “/” indicates that the antibody was not produced since the shown amino acid is the same as that of ACE910.

TABLE 2 FVIII cofactor function-substituting activity of modified bispecific antibodies produced by introducing amino acid substitutions into Q499/L404//J327/L404 (ACE910) po- mu- sition tation A I L M P V G N Q S T D E H K R F W Y L chain of anti-FIX(a) antibody was modified 24 K 1.96 1.51 2.22 2.05 0.91 1.47 1.42 1.18 2.01 2.17 1.50 1.54 1.81 2.06 / 2.63 0.07 1.04 1.48 25 A / 1.91 2.13 2.26 1.18 2.25 1.69 1.16 0.73 2.36 2.34 0.26 0.19 0.49 0.86 0.69 2.09 1.61 0.89 26 S 2.76 1.02 1.79 2.18 2.63 2.93 2.62 2.51 3.52 / 2.82 2.05 2.62 3.17 3.49 4.26 3.60 2.45 3.83 27 R 1.03 — 1.29 1.64 0.07 1.02 2.05 1.02 2.06 1.07 — 0.96 0.99 1.57 2.79 / 0.80 1.16 1.15 28 N 1.55 1.75 0.89 1.77 2.00 1.77 1.66 / 3.12 1.73 1.83 1.35 0.07 1.84 1.22 1.20 1.42 2.72 1.75 29 I 2.37 / 1.17 1.87 1.20 2.35 1.27 2.10 1.19 2.20 2.97 0.07 0.07 0.94 0.92 1.13 0.69 0.98 0.89 30 E 2.82 1.84 0.07 3.32 1.59 1.81 2.59 3.75 2.21 1.55 0.07 2.74 / 1.52 1.93 1.78 3.60 3.87 3.59 31 R 0.54 0.74 0.66 0.51 0.07 0.61 0.49 0.41 1.01 1.08 0.59 0.07 0.07 0.49 1.36 / 0.07 0.83 0.07 32 Q 1.17 0.84 1.58 1.96 0.07 1.07 2.26 1.70 / 1.21 0.93 0.97 1.05 1.28 3.28 3.23 — 1.00 2.03 33 L 1.65 1.96 / 1.62 0.07 1.71 0.82 1.16 2.10 1.89 1.96 1.01 1.29 1.15 1.01 0.31 1.30 0.07 0.68 34 A / 1.39 0.07 0.07 — 1.22 1.77 0.07 0.07 1.66 1.98 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 50 Q 1.42 1.00 1.18 1.15 0.15 1.32 1.08 0.63 / 1.27 0.07 0.96 1.05 1.23 1.43 1.71 1.06 1.16 0.07 51 A / — — — 0.31 0.96 1.16 — 0.36 1.08 1.26 0.25 0.07 0.77 — — — — — 52 S 0.92 0.99 0.94 0.77 0.07 1.06 1.06 0.90 1.00 / 1.11 0.68 0.89 1.08 0.07 1.11 0.95 1.04 1.03 53 R 0.79 1.24 1.15 1.28 0.91 0.97 0.95 0.84 0.86 0.98 0.92 0.44 0.38 0.94 1.10 / 1.45 0.97 1.05 54 K 1.26 1.38 1.22 1.24 1.14 1.12 1.28 0.53 1.27 1.31 1.09 1.01 0.91 1.21 / 1.27 1.46 1.45 1.47 55 E 1.24 1.39 1.18 1.23 1.10 1.13 1.14 1.28 0.07 0.07 1.21 1.25 / 1.06 1.04 0.94 1.10 0.98 0.07 56 S 1.15 1.18 1.07 1.14 1.11 1.28 1.22 1.08 1.12 / 1.16 1.02 1.07 1.16 1.46 1.20 1.21 1.21 1.12 89 Q 1.39 — 0.78 0.38 — 0.82 0.61 — / 1.08 1.01 0.52 1.06 0.40 0.68 0.20 — 0.94 1.08 90 Q 0.90 0.37 0.51 0.85 — 0.71 0.69 0.73 / 0.99 0.53 — 0.52 1.23 0.45 0.24 0.10 0.07 0.07 91 Y 0.07 0.07 0.07 0.12 0.07 0.11 0.13 0.14 0.07 0.07 0.07 0.13 0.07 0.26 0.11 0.07 0.92 0.60 / 92 S 0.99 1.07 0.72 0.92 0.22 0.56 0.94 1.12 0.79 / 0.76 0.85 0.57 1.19 1.13 1.10 0.94 0.66 0.99 93 D 0.96 0.07 1.06 1.12 0.63 1.20 0.93 1.34 1.34 1.26 1.12 / 0.84 1.14 1.28 1.53 1.33 0.96 1.32 94 P 0.68 0.74 0.40 0.63 / 0.65 0.50 0.32 0.31 0.39 0.21 0.07 0.07 0.44 0.10 0.12 0.12 0.07 0.29 95 P 0.86 0.56 0.62 0.61 / 0.94 1.14 0.75 0.55 0.94 0.90 0.84 0.82 0.80 1.91 0.82 0.44 0.53 0.36 96 L 1.27 0.78 / 1.16 1.19 0.93 1.32 1.00 1.07 1.10 0.99 0.44 0.07 0.91 0.63 0.31 1.10 1.17 0.07 97 T — 1.20 0.94 0.07 0.49 0.98 1.15 0.07 1.02 1.26 / 0.64 0.81 0.89 0.99 0.90 0.74 0.73 0.94 L chain of anti-FX antibody was modified 24 K 1.06 1.06 1.11 1.07 0.06 1.02 0.96 0.06 1.11 1.07 1.08 1.13 1.06 1.02 / 1.02 — 1.09 1.05 25 A / 0.68 0.67 0.59 0.87 0.73 0.06 0.98 0.53 1.18 1.05 0.22 0.49 0.31 — — 0.34 0.32 — 26 S 1.05 0.99 1.10 0.06 1.25 0.90 1.07 1.00 1.05 / 1.02 1.01 0.06 1.06 1.13 1.17 0.98 1.11 0.92 27 R 0.80 0.75 0.97 1.06 0.70 1.15 0.99 1.08 1.09 — 1.09 0.90 1.03 1.11 1.12 / 0.77 0.06 0.06 28 N 1.20 1.12 1.16 1.14 1.04 1.12 0.97 / 0.06 1.01 0.98 1.20 1.02 0.94 0.93 0.94 0.06 0.82 0.99 29 I 0.45 / 0.90 0.99 0.79 0.87 0.06 0.28 0.12 0.33 0.62 0.20 0.15 0.18 0.11 0.15 1.05 0.25 0.35 30 E 1.09 1.11 1.06 1.15 0.73 1.16 0.98 1.17 0.88 0.84 0.71 0.84 / 1.23 1.29 1.37 1.13 1.09 1.00 31 R 0.81 0.88 0.89 0.78 0.84 0.86 0.73 0.69 0.77 0.72 0.81 0.70 0.80 0.83 0.92 / 0.82 0.91 0.85 32 Q 0.89 0.25 0.84 1.04 0.76 0.35 1.09 0.83 / 1.14 0.69 0.74 0.59 0.79 1.29 1.31 0.67 0.74 0.83 33 L 1.02 0.97 / 1.01 0.38 0.87 0.80 1.06 0.94 0.86 1.10 0.06 — 0.85 0.21 — 1.16 0.40 0.30 34 A / 1.13 0.71 0.72 — 1.06 1.08 0.69 1.00 1.03 0.82 0.38 0.98 0.88 0.06 0.06 0.53 0.29 0.42 50 Q 0.96 0.92 0.91 0.87 0.82 0.84 0.91 0.74 / 0.91 0.82 0.82 0.85 0.93 1.29 1.34 1.01 0.91 1.04 51 A / 1.02 — 1.19 0.74 1.04 0.06 — 0.84 4.16 0.94 0.78 0.86 0.88 — — 0.99 — — 52 S 1.17 1.21 1.27 0.98 0.70 1.12 0.97 1.07 0.97 / 1.03 1.00 0.79 1.07 0.73 1.01 1.10 0.96 0.93 53 R 0.76 0.72 0.86 0.81 0.77 0.89 0.91 0.79 0.78 0.80 0.82 0.66 0.72 0.83 1.02 / 0.75 0.76 0.74 54 K 0.91 0.90 1.15 1.22 0.99 0.96 0.57 0.92 1.01 0.90 0.85 0.80 0.91 0.99 / 0.90 1.02 0.81 0.98 55 E 1.37 1.37 — 1.14 1.17 1.10 1.29 1.31 0.06 1.21 1.13 1.03 / 1.26 1.23 1.37 1.18 — 1.30 56 S 0.99 0.88 0.86 0.83 0.87 0.81 0.91 0.86 0.91 / 0.90 0.85 0.90 0.88 1.15 0.96 0.88 0.88 0.86 89 Q 0.90 — 0.92 1.04 — 1.02 0.78 — / 0.79 0.96 — 1.00 0.77 0.93 0.06 — 0.50 0.68 90 Q 0.23 0.21 0.31 — 0.27 0.17 0.12 0.58 / 0.36 0.17 — 0.64 0.45 — 0.46 — 0.09 0.19 91 Y 0.06 0.26 0.20 0.17 0.10 0.13 0.06 0.14 — 0.06 0.11 0.06 0.06 0.09 0.23 0.21 0.15 — / 92 S 0.79 0.74 0.87 0.93 0.44 0.88 0.82 0.88 0.81 / 0.80 0.90 0.86 0.98 1.20 1.14 0.90 0.93 0.82 93 D 1.11 1.04 1.17 1.10 0.88 0.94 1.01 0.95 1.01 0.96 1.13 / 1.04 1.15 1.13 1.26 1.06 1.17 1.12 94 P 1.03 1.21 1.02 1.08 / 1.09 0.69 0.68 — 1.20 0.97 0.41 0.28 1.07 0.63 0.60 0.51 0.64 1.23 95 P 0.26 0.22 0.36 0.29 / 0.23 0.17 0.31 0.30 0.33 0.45 0.24 0.23 0.31 0.37 0.24 0.29 0.28 0.21 96 L 1.03 1.02 / 0.69 — 0.96 — 0.47 0.93 0.87 1.09 0.31 0.43 0.41 0.15 0.06 0.76 0.85 0.84 97 T — 1.18 0.56 0.80 0.68 1.12 0.54 0.58 0.68 1.23 / 0.65 0.77 0.61 1.12 — 0.67 0.52 —

Example 2 Obtaining Novel L Chains that are Compatible with Each H Chain of ACE910

As indicated in Example 1, FVIII cofactor function-substituting activity was able to be improved by comprehensively modifying amino acids in L404, which is the commonly shared L chain of ACE910. As another method of improving the FVIII cofactor function-substituting activity of ACE910, a conceivable method is that of obtaining from human antibody libraries novel L chains having sequences completely different from that of the commonly shared L chain, which are to be paired with the respective H chains of the anti-FIX(a) antibody and the anti-FX antibody (Q499 and J327).

With reference to methods known to those skilled in the art, specifically the method of Biochemical and Biophysical Research Communications, (2000), 275, 2: 553-557 and such, the present inventors newly produced a library of antibodies in which the L chain portion of the ACE910 antibody has been substituted with that from a human L chain library, then performed panning operations on biotinylated human FIXa or biotinylated human FX, and thereby succeeded in obtaining antibodies comprising novel L chains and having FVIII cofactor function-substituting activity.

As a result, for bispecific antibodies having FVIII cofactor function-substituting activity, the followings were discovered as the anti-human FIX(a) antibody L chains: QNK131 (heavy chain SEQ ID NO: 45 and light chain SEQ ID NO: 13), QNK284 (heavy chain SEQ ID NO: 45 and light chain SEQ ID NO: 14), QNK315 (heavy chain SEQ ID NO: 45 and light chain SEQ ID NO: 15), QNL182 (heavy chain SEQ ID NO: 45 and light chain SEQ ID NO: 16), QNL492 (heavy chain SEQ ID NO: 45 and light chain SEQ ID NO: 17), and QNL576 (heavy chain SEQ ID NO: 45 and light chain SEQ ID NO: 18); and, as the anti-human FX antibody L chains: JNK131 (heavy chain SEQ ID NO: 46 and light chain SEQ ID NO: 19), JNK163 (heavy chain SEQ ID NO: 46 and light chain SEQ ID NO: 20), JNK252 (heavy chain SEQ ID NO: 46 and light chain SEQ ID NO: 21), JNK263 (heavy chain SEQ ID NO: 46 and light chain SEQ ID NO: 22), JNK339 (heavy chain SEQ ID NO: 46 and light chain SEQ ID NO: 23), JNK348 (heavy chain SEQ ID NO: 46 and light chain SEQ ID NO: 24), JNK351 (heavy chain SEQ ID NO: 46 and light chain SEQ ID NO: 25), JNK360 (heavy chain SEQ ID NO: 46 and light chain SEQ ID NO: 26), JNK378 (heavy chain SEQ ID NO: 46 and light chain SEQ ID NO: 27), JNK382 (heavy chain SEQ ID NO: 46 and light chain SEQ ID NO: 28), JNL036 (heavy chain SEQ ID NO: 46 and light chain SEQ ID NO: 29), JNL072 (heavy chain SEQ ID NO: 46 and light chain SEQ ID NO: 30), JNL095 (heavy chain SEQ ID NO: 46 and light chain SEQ ID NO: 31), JNL176 (heavy chain SEQ ID NO: 46 and light chain SEQ ID NO: 32), JNL208 (heavy chain SEQ ID NO: 46 and light chain SEQ ID NO: 33), JNL224 (heavy chain SEQ ID NO: 46 and light chain SEQ ID NO: 34), JNL260 (heavy chain SEQ ID NO: 46 and light chain SEQ ID NO: 35), JNL056 (heavy chain SEQ ID NO: 46 and light chain SEQ ID NO: 36), JNL059 (heavy chain SEQ ID NO: 46 and light chain SEQ ID NO: 37), JNL226 (heavy chain SEQ ID NO: 46 and light chain SEQ ID NO: 38), JNL250 (heavy chain SEQ ID NO: 46 and light chain SEQ ID NO: 39), JNL263 (heavy chain SEQ ID NO: 46 and light chain SEQ ID NO: 40), and JNL281 (heavy chain SEQ ID NO: 46 and light chain SEQ ID NO: 41).

Various bispecific antibodies comprising these novel L chains were expressed and purified by methods known to those skilled in the art. The prepared antibodies are shown in Table 3 (clone names, heavy chain SEQ ID NOs, and anti-FIX(a) light chain SEQ ID NOs or the anti-FX light chain SEQ ID NOs are shown). The novel L chain was used for only one arm and L404 which is the commonly shared L chain of ACE910 was used for the other arm. The results of measuring the FVIII cofactor function-substituting activities of the various purified bispecific antibodies are shown in FIG. 1. All of the novel L chain-comprising bispecific antibodies were confirmed to have FVIII cofactor function-substituting activity.

TABLE 3 Prepared bispecific antibodies having novel L chains Clone name Heavy chain SEQ ID NO Light chain SEQ ID NO QNK131 45 13 QNK284 45 14 QNK315 45 15 QNL182 45 16 QNL492 45 17 QNL576 45 18 JNK131 46 19 JNK163 46 20 JNK252 46 21 JNK263 46 22 JNK339 46 23 JNK348 46 24 JNK351 46 25 JNK360 46 26 JNK378 46 27 JNK382 46 28 JNL036 46 29 JNL072 46 30 JNL095 46 31 JNL176 46 32 JNL208 46 33 JNL224 46 34 JNL260 46 35 JNL056 46 36 JNL059 46 37 JNL226 46 38 JNL250 46 39 JNL263 46 40 JNL281 46 41

Example 3 Production of Variants of the Novel L Chain-Comprising Bispecific Antibodies

To improve the FVIII cofactor function-substituting activity of novel L chain-comprising bispecific antibodies obtained in Example 2, amino acid substitution modifications were introduced into QNK131 (light chain SEQ ID NO: 13) which is a novel L chain of an anti-FIX(a) antibody, to obtain QAL187 (light chain SEQ ID NO: 42) and QAL201 (light chain SEQ ID NO: 43). Similarly, amino acid substitution modifications were introduced into JNL095 (light chain SEQ ID NO: 31) which is a novel L chain of an anti-FX antibody, to obtain JYL280 (light chain SEQ ID NO: 44). In one example, a bispecific antibody (Q499/QAL201/J327/JYL280) composed of an anti-FIX(a) antibody comprising heavy chain Q499 and light chain QAL201 and an anti-FX antibody comprising heavy chain J327 and light chain JYL280 was expressed and purified by a method known to those skilled in the art, and the result of measuring its FVIII cofactor function-substituting activity is shown in FIG. 2.

Example 4 Production of Variants of the H Chain of the Novel L Chain-Comprising Bispecific Antibodies

Using novel L chains (QAL187 for the anti-FIX(a) antibody, and JYL280 for the anti-FX antibody) with improved FVIII cofactor function-substituting activities, the inventors comprehensively introduced amino acid mutations to Q499 and J327, and performed large-scale screening of FVIII cofactor function-substituting activity to discover amino acid substitutions that improve the FVIII cofactor function-substituting activity.

The heavy chain and light chain of the anti-FIX(a) antibody side were fixed to Q499 and QAL201, respectively, and the light chain of the anti-FX antibody side was fixed to JYL280 to produce substitution variants in which all amino acids in CDRs of J327 have been modified to amino acids other than cysteine. The heavy chain and light chain of the anti-FX antibody side were fixed to J327 and JYL280, respectively, and the light chain of the anti-FIX(a) antibody side was fixed to QAL187 to produce substitution variants in which all amino acids in CDRs of Q499 have been modified to all amino acids other than cysteine. Expression and purification of the bispecific antibodies were carried out by methods known to those skilled in the art.

FVIII cofactor function-substituting activities were evaluated by methods known to those skilled in the art by using the various purified bispecific antibodies.

As a result, several amino acid substitutions indicated in Table 4 below were found to be able to improve the FVIII cofactor function-substituting activity as compared with the parent bispecific antibody Q499/QAL187//J327/JYL280 or Q499/QAL201//J327/JYL280. Table 4 shows the mutated positions indicated by Kabat numbering (the number in the “position” column on the far left), the amino acids in the parent antibody (amino acids (one letter code) in the second column from the left), amino acids after the mutation (amino acids (one letter code) in the uppermost row), and the specific activity (each numerical value in the table) relative to ACE910 (purified antibody). In Table 4, (−) indicates that the expression level of the antibody was low, and “/” indicates that the antibody was not produced since the shown amino acid is the same as that of the parent antibody.

TABLE 4 po- mu- sition tation A I L M P V G N Q S T D E H K R F W Y FVIII cofactor function-substituting activity of modified bispecific antibodies produced by introducing amino acid substitutions into Q499 of Q499/QAL187//J327/JYL280  31 Y 0.4 0.0 — — 0.0 0.0 0.0 0.0 — 1.0 0.0 0.0 0.0 3.5 0.0 0.0 0.0 10.0 /  32 Y 0.6 1.3 0.0 0.0 0.0 1.5 — 1.4 2.0 0.0 6.2 2.2 0.1 0.9 1.2 0.0 6.1 0.6 /  33 D — — — 0.0 — 0.1 — 0.0 — — 0.2 / 0.1 — — — — — —  34 I 1.2 / 10.7 8.9 0.0 0.0 2.4 — 8.0 0.2 2.9 0.2 1.5 0.0 1.1 0.0 41.8 13.2 0.0  35 Q 0.4 4.6 76.2 — 39.0 45.7 73.9 0.0 / 22.8 56.0 0.0 0.1 0.1 1.4 0.0 0.5 0.0 0.1  50 S 5.4 1.0 0.9 4.1 0.0 — 2.8 6.0 0.0 / 0.0 3.3 0.0 — 0.2 0.0 1.2 3.3 1.3  51 I 0.0 / — 0.0 0.0 2.3 0.0 0.0 — 0.3 0.0 0.0 — — 0.0 0.0 — 0.0 0.0  52 S 1.1 4.8 0.0 0.0 0.0 3.8 1.0 9.9 8.1 / — 13.1 0.0 34.0 0.1 — 26.0 8.2 2.0  52a P 0.4 — 0.7 — / 0.1 4.9 0.0 1.6 0.0 0.0 0.0 0.3 0.0 — 0.0 0.0 0.1 1.3  53 S 0.9 0.1 0.3 0.0 0.0 0.0 1.3 0.0 0.0 / 1.4 0.1 0.0 1.3 0.0 0.2 0.3 0.0 0.2  54 G 1.3 0.0 0.0 — 0.0 0.0 / 0.0 0.1 1.2 — 0.0 0.0 — 0.0 — — 0.0 0.1  55 Q 0.0 0.0 — 0.0 0.0 0.2 3.9 0.0 / — 0.0 — 0.0 4.1 — 7.4 0.0 0.0 0.0  56 S — 0.0 1.9 13.1 0.2 0.0 3.2 0.0 — / 0.2 1.1 0.6 6.2 0.8 1.9 0.0 — 25.8  57 T 2.7 2.1 3.0 3.1 0.2 1.7 — 0.9 3.8 2.6 / 0.2 1.6 1.9 4.2 0.7 2.3 1.5 2.0  58 Y — 1.7 1.9 0.4 — 1.1 0.2 5.0 0.6 0.6 14.7 3.8 — 3.8 3.6 0.2 4.5 1.5 /  59 Y 0.0 0.5 — 1.0 0.0 1.0 0.0 0.1 — — — 0.0 1.5 — 0.0 0.0 1.0 0.4 /  60 R 3.7 4.1 3.4 3.9 1.2 3.4 2.8 — 4.1 3.5 2.3 — — 4.1 3.7 / 4.0 0.0 —  61 R 1.8 1.5 1.5 1.6 1.7 1.6 2.0 2.1 2.2 0.0 1.9 1.1 0.7 3.7 — / 1.7 1.1 —  62 E 3.3 1.1 1.8 1.3 3.4 1.4 4.2 4.6 4.4 5.2 4.5 6.0 / 3.0 3.0 0.0 — 1.6 0.0  63 V 2.9 2.5 3.1 1.3 1.0 / 0.1 0.5 0.1 0.4 1.1 0.5 0.1 3.5 0.1 0.0 4.0 0.5 6.6  64 K 1.2 1.5 1.3 1.2 0.0 1.1 1.1 1.0 1.2 1.3 1.1 0.4 0.4 1.2 / 4.8 0.0 0.8 1.1  65 G 2.1 4.4 3.0 2.9 0.2 1.2 / 3.5 20.4 2.3 3.1 3.8 2.5 4.1 1.4 2.7 2.5 3.5 3.3  95 R 0.0 0.4 0.0 — 0.1 0.2 0.0 0.0 1.1 0.0 0.1 — — — 1.2 / 0.0 0.0 0.0  96 T 5.5 0.5 0.4 1.6 0.1 1.3 — 3.3 15.0 15.0 / 4.2 1.5 1.6 0.5 0.3 1.3 2.1 2.6  97 G — 0.1 0.0 0.3 3.1 0.4 / 1.4 0.7 2.1 0.4 11.6 1.8 1.2 0.2 0.1 1.0 1.9 1.1  98 R 4.5 9.0 9.5 5.8 2.2 4.1 5.7 7.7 7.6 5.7 8.1 3.7 4.1 5.8 4.0 / 5.8 3.9 8.2  99 E 3.4 10.4 7.0 5.5 5.8 7.0 1.7 3.0 4.3 4.0 4.8 3.4 / 4.2 2.2 2.0 3.3 1.8 3.6 100 Y 1.6 5.0 1.6 3.3 2.7 3.4 1.2 3.2 1.5 1.3 1.6 3.0 1.5 3.2 1.9 2.2 2.1 3.6 / 100a G 3.4 1.1 5.1 5.8 1.4 0.0 / 2.7 — 1.7 1.0 2.6 0.9 2.5 6.5 7.1 2.1 2.0 2.7 100b G 5.3 1.0 0.4 0.4 0.8 0.9 / 1.1 1.0 2.3 1.1 1.4 1.2 0.6 — 0.2 0.0 0.0 0.2 100c G 0.2 0.0 0.0 0.0 0.1 0.0 / 1.1 0.0 0.4 0.0 0.0 0.0 0.0 — 0.0 0.0 0.0 0.0 100d W 0.0 0.5 0.3 0.3 0.3 0.6 0.0 0.0 0.0 0.1 0.1 0.0 0.0 1.4 0.0 0.0 10.3 / 3.6 100e Y 3.1 4.5 3.7 3.0 0.4 4.9 1.0 5.1 3.2 3.8 5.2 1.7 1.5 9.4 1.1 — 3.1 1.5 / 100f F 11.7 24.9 7.6 8.6 7.8 23.2 — 18.6 6.3 8.0 11.3 1.3 2.0 0.7 0.0 0.6 / 0.1 0.7 101 D 3.2 4.1 2.1 1.8 8.6 3.9 2.3 3.2 2.6 3.1 2.9 / 2.7 2.4 2.3 2.5 2.1 3.0 2.5 102 Y 4.4 4.1 4.6 4.1 4.6 5.8 6.3 4.3 4.3 2.6 5.9 8.6 6.5 4.6 3.6 3.2 3.9 3.5 / FVIII cofactor function-substituting activity of modified bispecific antibodies produced by introducing amino acid substitutions into J327 of Q499/QAL201//J327/JYL280  31 D — 8.9 13.0 14.2 — 11.0 15.5 14.7 14.6 15.3 12.5 / 14.0 17.4 12.6 11.2 13.7 13.3 13.6  32 N 10.7 0.4 0.8 5.6 0.3 1.9 3.3 / 4.1 12.5 4.2 0.2 0.8 5.2 3.1 3.5 4.4 1.5 4.3  33 N 0.4 1.8 2.8 1.7 0.0 1.2 1.2 / 1.8 3.4 0.1 0.2 0.0 0.0 1.5 0.1 0.0 0.2 0.0  34 M 2.6 11.0 12.1 / — 12.4 — — — — 1.6 — — 7.8 — — 7.4 7.3 3.4  35 D 3.4 4.2 3.1 6.6 — 5.0 3.5 13.1 11.4 5.0 0.3 / 11.4 6.0 — — 3.5 4.8 2.3  50 D 3.1 0.3 0.2 0.5 — 2.4 1.2 4.1 1.8 3.9 1.7 / 1.3 0.1 0.1 0.0 0.6 0.0 0.1  51 I 10.2 / 10.3 10.2 — 12.8 1.6 — 9.5 11.5 12.7 0.6 0.7 — 3.1 0.1 4.8 0.7 4.7  52 N 1.8 0.1 0.1 0.1 0.1 0.1 0.6 / 0.7 2.9 0.0 0.1 0.5 3.5 0.0 0.1 0.1 0.0 0.0  52a T 6.0 7.7 4.2 3.9 7.2 9.0 3.2 — 0.9 11.9 / 0.1 0.0 1.0 0.3 0.1 1.5 4.0 0.7  53 R 3.7 7.4 5.0 5.3 3.0 6.9 3.9 12.4 6.0 3.8 0.3 1.4 1.9 7.2 9.8 / 7.4 8.9 5.8  54 S 12.8 4.3 3.6 6.2 0.1 8.0 10.8 5.3 5.4 / 6.3 2.7 1.1 5.8 3.1 4.7 10.1 16.0 12.2  55 G 11.0 6.2 8.3 8.7 3.1 8.0 / 2.7 11.0 12.1 9.2 4.4 6.6 10.4 7.0 7.9 7.6 7.3 6.7  56 G 8.3 11.2 11.4 11.6 — 8.8 / 11.3 13.3 13.3 13.8 4.6 3.2 8.4 10.2 12.3 10.4 13.7 10.3  57 S 11.9 10.9 6.5 12.2 12.1 12.9 13.7 10.6 12.9 / 10.9 6.9 0.7 2.5 0.1 0.3 0.8 3.0 0.1  58 I 5.8 / 7.7 3.9 — 3.7 1.3 6.3 7.2 2.7 11.3 0.1 2.7 3.3 7.5 4.2 4.8 2.6 5.5  59 Y 3.2 11.8 5.9 3.7 0.4 1.3 1.7 5.9 7.8 7.2 9.4 6.7 6.0 12.7 4.0 3.2 4.9 6.6 /  60 N 11.0 9.0 10.4 10.2 5.0 9.6 5.2 / 6.1 2.2 1.1 10.7 4.4 2.2 4.6 10.1 9.8 10.0 11.7  61 E 17.6 12.5 10.8 6.4 6.8 2.3 7.5 2.6 11.1 12.0 7.9 3.6 / 5.3 9.6 6.9 8.1 7.7 6.9  62 E 9.5 9.9 10.6 11.1 9.3 11.6 13.6 13.0 15.2 4.2 2.9 — / 9.3 9.8 12.6 10.0 13.7 10.9  63 F 12.5 11.3 6.3 12.1 — 12.2 13.1 12.6 10.2 12.3 — 11.3 6.3 15.9 7.8 6.2 / 16.7 16.3  64 Q 16.2 14.7 11.8 16.0 9.7 14.8 15.0 15.0 / 15.8 15.3 14.4 16.2 13.7 14.2 12.4 15.7 13.9 15.5  65 D 14.3 — 12.6 15.5 16.1 — 11.5 15.0 15.4 15.6 14.9 / 14.2 14.8 12.9 14.5 14.9 12.4 14.8  95 R 0.2 0.6 1.6 1.0 0.7 1.0 0.1 0.0 0.8 0.1 0.2 0.0 — 1.2 5.7 / 0.0 0.4 0.3  96 K 3.0 2.0 1.8 4.3 0.0 3.6 1.1 1.0 2.3 1.2 1.7 0.0 1.0 1.7 / 15.7 1.9 1.9 1.4  97 S 14.8 2.2 1.6 5.2 1.4 3.7 10.2 5.4 6.5 / 7.6 2.9 3.9 7.2 3.5 — 3.2 1.5 3.2  98 Y 2.2 1.1 1.9 7.3 0.1 1.4 1.4 1.3 1.9 1.9 1.0 0.2 0.2 8.8 1.5 7.9 6.9 4.2 /  99 G 4.6 — — 5.2 0.0 0.5 / 7.6 7.6 6.8 1.7 8.8 3.7 6.1 5.7 7.2 6.3 3.7 4.0 100 Y 12.6 8.3 14.5 10.8 6.2 10.9 9.3 12.6 12.9 12.0 11.5 10.9 7.8 12.0 12.8 12.2 13.9 6.5 / 100a Y 5.9 2.7 5.7 3.5 1.3 7.0 2.8 8.9 3.9 7.1 7.7 3.3 3.9 14.7 2.4 6.7 13.4 11.6 / 100b L 4.1 10.6 / 12.9 0.6 7.2 0.0 4.8 3.9 1.9 3.1 1.5 0.3 8.0 0.8 0.2 14.8 6.2 11.2 101 D 6.5 5.2 5.4 5.3 6.7 6.8 6.7 6.7 7.0 6.8 6.2 / 7.0 3.4 4.4 4.1 — — — 102 E 12.1 11.7 10.8 11.7 6.9 13.3 12.6 11.2 11.3 9.4 10.2 12.5 / 10.3 10.9 10.5 10.5 12.7 12.9

INDUSTRIAL APPLICABILITY

Light chain amino acid substitutions that improve the FVIII cofactor function-substituting activity of ACE910 (Emicizumab), novel light chains showing FVIII cofactor function-substituting activity, and heavy chain amino acid substitutions that improve the FVIII cofactor function-substituting activity of novel light chain-comprising bispecific antibodies were found by the present invention. These amino acid substitutions and novel light chains are useful for producing bispecific antibodies having FVIII cofactor function-substituting activities that are superior to that of ACE910 (Emicizumab). 

1. A polypeptide comprising an antibody light chain variable domain, wherein the polypeptide comprises an antibody light chain variable domain having the light chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, or an antibody light chain variable domain having the amino acid sequence of SEQ ID NO: 47, in which one or more amino acids selected from the group consisting of K24, A25, S26, R27, N28, I29, E30, R31, Q32, L33, A34, Q50, A51, S52, R53, K54, E55, S56, Q89, Q90, Y91, S92, D93, P94, P95, L96, and197, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine.
 2. A bispecitic antibody which recognizes FIX and/or FIXa, and FX, wherein the antibody comprises an antibody light chain variable domain having the light chain CDR 1, 2, and 3 amino acid sequences of SEQ NOs: 7, 8, and 9, respectively, or an antibody light chain variable domain having the amino acid sequence of SEQ ID NO: 47, in which one or more amino acids selected from the group consisting of K24, A25, S26, R27, N28, 129, E30, R31, Q32, L33, A34, Q50, A51, S52, R53, K54, E55, S56, Q89, Q90, Y91, S92, D93, P94, P95, L96, and197, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine.
 3. A bispecific antibody which recognizes FIX and/or FIXa, and FX, wherein a first polypeptide and a third polypeptide form a pair and a second polypeptide and a fourth polypeptide form a pair, wherein the first polypeptide comprises the antibody heavy chain variable domain amino acid sequence of SEQ ID NO: 45 and the second polypeptide comprises the antibody heavy chain variable domain amino acid sequence of SEQ ID NO: 46, wherein either the third polypeptide or the fourth polypeptide comprises the antibody light chain variable domain amino acid sequence of SEQ If) NO: 47 and the other polypeptide comprises an antibody light chain variable domain having the light chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, or an antibody light chain variable domain having the amino acid sequence of SEQ ID NO: 47, and wherein in the other polypeptide one or more amino acids selected from the group consisting of K24, A25, S26, R27, N28, 129, E30, R31, Q32, L33, A34, Q50A51, S52, R53, K54, E55, S56, Q89, Q90, Y91, S92, D93, P94, P95, L96, and 197, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine.
 4. A polypeptide comprising an antibody heavy chain variable domain, wherein the polypeptide comprises an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, or an antibody heavy chain variable domain having the amino acid sequence of SEQ ID NO: 45, in which one or more amino acids selected from the group consisting of Y31, Y32, D33, 134, Q35, S50, I51, S52, P52a, S53, G54, Q55, S56, T57, Y58, Y59, R60, R61, E62, V63, K64, G65, R95, T96, G97, R98, E99, Y100, G100a, G100b, G100c, W100d, Y100e, F100f, D101, and Y102, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine.
 5. A bispecific antibody which recognizes FIX and/or FIXa, and FX, wherein the antibody comprises an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, or comprises an antibody heavy chain variable domain having the amino acid sequence of SEQ ID NO: 45, in which one or more amino acids selected from the group consisting of Y31, Y32, D33,
 134. Q35, S50, I51, S52, P52a, S53, G54, Q55, S56, T57, Y58, Y59, R60, R61, E62, V63, K64, G65, R95, T96, G97, R98, E99, Y100, G100a, G100b, G100c, W100d, Y100e, F100f, D101, and Y102, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine.
 6. A polypeptide comprising an antibody heavy chain variable domain, wherein the polypeptide comprises an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively, or an antibody heavy chain variable domain having the amino acid sequence of SEQ ID NO: 46, in which one or more amino acids selected from the group consisting of D31, N32, N33, M34, D35, D50, I51, N52, T52a, R53, S54, G55, G56, S57, 158, Y59, N60, E61, E62, F63, Q64, D65, R95, K96, S97, Y98, G99, Y100, Y100a, L100b, D101, and E102, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine.
 7. A bispecific antibody which recognizes FIX and/or FIXa, and FX, wherein the antibody comprises an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively, or comprises an antibody heavy chain variable domain having the amino acid sequence of SEQ ID NO: 46, in which one or more amino acids selected from the group consisting of D31, N32, N33, M34, D35, D50, I51, N52, T52a, R53, S54, G55, G56, S57, I58, Y59, N60, E61, E62, F63, Q64, D65, R95, K96, S97, Y98, G99, Y100, Y100a, L100b, D101, and E102, numbered according to the Kabat numbering system, have been substituted with an arbitrary amino acids other than cysteine.
 8. A bispecific antibody which recognizes FIX and/or FIXa, and FX, wherein a first polypeptide and a third polypeptide form a pair and a second polypeptide and a fourth polypeptide form a pair, wherein the second polypeptide comprises the antibody heavy chain variable domain amino acid sequence of SEQ ID NO: 46, the third polypeptide comprises the antibody light chain amino acid sequence of SEQ ID NO: 42, and the fourth polypeptide comprises the antibody light chain amino acid sequence of SEQ ID NO: 44, and the first polypeptide comprises an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, or an antibody heavy chain variable domain having the amino acid sequence of SEQ ID NO: 45, and wherein in the first polypeptide one or more amino acids selected from the group consisting of Y31, Y32, D33, I34, Q35, S50, I51, S52, P52a, S53, G54, Q55, S56, T57, Y58, Y59, R60, R61, E62, V63, K64, G65, R95, T96, G97, R98, E99, Y100, G100a, G100b, G100c, W100d, Y100e, F100f, D101, and Y102, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine.
 9. A bispecific antibody which recognizes FIX and/or FIXa, and FX, wherein a first polypeptide and a third polypeptide form a pair and a second polypeptide and a fourth polypeptide form a pair, wherein the first polypeptide comprises the antibody heavy chain amino acid sequence of SEQ ID NO: 45, the third polypeptide comprises the antibody light chain amino acid sequence of SEQ ID NO: 43, and the fourth polypeptide comprises the antibody light chain amino acid sequence of SEQ ID NO: 44, and the second polypeptide comprises an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ NOs: 4, 5, and 6, respectively, or an antibody heavy chain variable domain having the amino acid sequence of SEQ ID NO: 46, and wherein in the second polypeptide one or more amino acids selected from the group consisting of D31, N32, N33, M34, D35, D50, 151, T52a, N52, R53, S54, G55, G56, S57, 158, Y59, N60, E61, E62, F63, Q64, D65, R95, K96, S97, Y98, G99, Y100, Y100a, L100b, D101, and E102, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine.
 10. An antibody light chain, which comprises any one of the amino acid sequences selected from the following (a1) to (a6), (b1) to (b23), and (c1) to (c3): (a1) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 13 (QNK131); (a2) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 14 (QNK284); (a3) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 15 (QNK315); (a4) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 16 (QNL182); (a5) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 17 (QNL492); (a6) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 18 (QNL576); (b1) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 19 (JNK131); (b2) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 20 (JNK163); (b3) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 21 (JNK252); (b4) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 22 (JNK263); (b5) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 23 (JNK339); (b6) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 24 (JNK348); (b7) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 25 (JNK351); (b8) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 26 (JNK:360); (b9) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 27 (JNK378); (b10) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 28 (JNK382); (b11) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 29 (JNL036); (b12) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 30 (JNL072); (b13) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 31 (JNL095); (b14) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 32 (JNL176); (b15) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 33 (JNL208); (b16) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 34 (JNL224); (b17) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 35 (JNL260); (b18) an antibody light chain comprising the amino acid sequence of SEQ NO: 36 (JNL056); (b19) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 37 (JNL059); (b20) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 38 (JNL226); (b21) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 39 (JNL250); (b22) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 40 (JNL263); (b23) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 41 (JNL281); (c1) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 42 (QAL187); (c2) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 43 (QAL201); and (c3) an antibody light chain comprising the amino acid sequence of SEQ ID NO: 44 (JYL280).
 11. A bispecific antibody which recognizes FIX andlor FIXa, and FX, wherein a first polypeptide (d) and a third polypeptide (f) form a pair, a second polypeptide (e) and a fourth polypeptide (g) form a pair, and wherein each of the polypeptides is the following polypeptide: (d) the first polypeptide is (d1) or (d2): (d1) a polypeptide which comprises an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ NOs: 1, 2, and 3, respectively, or an antibody heavy chain variable domain having the amino acid sequence of SEQ ID NO: 45; (d2) a polypeptide which comprises an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, or an antibody heavy chain variable domain having the amino acid sequence of SEQ ID NO: 45, in which one or more amino acids selected from the group consisting of Y31, Y32, D33, I34, Q35, S50, I51, S52, P52a, S53, G54, Q55, S56, T57, Y58, Y59, R60, R61, E62, V63, K64, G65, R95, T96, G97, R98, E99, Y100, G100a, G100b, G100c, W100d, Y100e, F100f, D101, and Y102, numbered according to the Kabat numbefing system, have been substituted with arbitrary amino acids other than cysteine; (e) the second polypeptide is (e1) or (e2): (e1) a polypeptide which comprises an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively, or an antibody heavy chain variable domain having the amino acid sequence of SEQ ID NO: 46; (e2) a polypeptide Which comprises an antibody heavy chain variable domain having the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively, or an antibody heavy chain variable domain having the amino acid sequence of SEQ ID NO: 46, in which one or more amino acids selected from the group consisting of D31, N32, N33, M34, D35, D50, I51, 152a, N52, R53, S54, G55, G56, S57, I58, Y59, N60, E61, E62, F63, Q64, D65, R95, K96, S97, Y98, G99, Y100, Y100a, L100b, D101, and E102, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine; (f) the third polypeptide is (f1), (f2), or (f3): (f1) a polypeptide which comprises an antibody light chain variable domain having the light chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, or an antibody light chain variable domain having the amino acid sequence of SEQ ID NO: 47; (f2) a polypeptide which comprises an antibody light chain variable domain having the light chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, or an antibody light chain variable domain having the amino acid sequence of SEQ ID NO: 47, in which one or more amino acids selected from the group consisting of K24, A25, S26, R27, N28, I29, E30, R31, Q32, I33, A34, Q50, A51, S52, R53, K54, E55, S56, Q89, Q90, Y91, S92, D93, P94, P95, L96, and T97, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine; (f3) the polypeptide of any one of (a1) to (a6) and (c1) to (c2) described in claim 10; and (g) the fourth polypeptide is (g1), (g2), or (g3): (g1) a polypeptide which comprises an antibody light chain variable domain having the light chain CDR 1, 2, and 3 amino acid sequences of SEQ NOs: 7, 8, and 9, respectively, or an antibody light chain variable domain having the amino acid sequence of SEQ ID NO: 47; (g2) a polypeptide which comprises an antibody light chain variable domain having the light chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, or an antibody light chain variable domain having the amino acid sequence of SEQ ID NO: 47, in which one or more amino acids selected from the group consisting of K24, A25, S26, R27, N28, I29, E30, R31, Q32, L33, A34, Q50, A51, S52, R53, K54, E55, S56, Q89, Q90, Y91, S92, D93, P94, P95, L96, and T97, numbered according to the Kabat numbering system, have been substituted with arbitrary amino acids other than cysteine; (g3) the polypeptide of any one of (b1) to (b23), and (c3) described in claim
 10. 12. A method of producing a variant of Emicizumab, which comprises the following step (a): (a) performing one or more of the following substitutions (i) to (iii), wherein the numbering is according to the Kabat numbering system: (i) substitution of one or more amino acids selected from the group consisting of K24, A25, S26, R27, N28, 129, E30, R31, Q32, L33, A34, Q50, A51, S52, R53, K54, E55, S56, Q89, Q90, Y91, S92, D93, P94, P95, L96, and197, in an antibody light chain variable domain comprising the light chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively; (ii) substitution of one or more amino acids selected. from the group consisting of Y31, Y32, D33, 134, Q35, S50, I51, S52, P52a, S53, G54, Q55, S56, T57, Y58, Y59, R60, R61, E62, V63, K64, G65, R95, T96, G97, R98, E99, Y100, G100a, G100b, G100c, W100d, Y100e, F100f, D101, and Y102, in an antibody heavy chain variable domain comprising the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ NOs: 1, 2, and 3, respectively; and (iii) substitution of one or more amino acids selected from the group consisting of D31, N32, N33, M34, D35, D50, I51, T52a, N52, R53, S54, G55, G56, S57, I58, Y59, N60, E61, E62, F63, Q64, D65, R95, K96, S97, Y98, G99, Y100, Y100a, L100b, D101, and E102, in an antibody heavy chain variable domain comprising the heavy chain CDR 1, and 3 amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.
 13. A method of isolating a variant of Emicizumab, which comprises the steps of (a) to (c) below: (a) producing a variant of Emicizumab by performing one or more of the following (i) to (iii), wherein the numbering is according to the Kabat numbering system: (i) substitution of one or more amino acids selected from the group consisting of K24, A25, S26, R27, N28, I29, E30, R31, Q32, L33, A34, Q50, A51, S52, R53, K54, E55, S56, Q89, Q90, Y91, S92, D93, P94, P95, L96, and T97, in an antibody light chain variable domain comprising the light chain CDR I, 2, and 3 amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively; (ii) substitution of one or more amino acids selected from the group consisting of Y31, Y32, D33, 134, Q35, S50, I51, S52, P52a, S53, G54, Q55, S56, T57, Y58, Y59, R60, R61, E62, V63, K64, G65, R95, T96, G97, R98, E99, Y100, G100a, G100b, G100c, W100d, Y100e, F100f, D101, and Y102, in an antibody heavy chain variable domain comprising the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively; and (iii) substitution of one or more amino acids selected from the group consisting of D31, N32, N33, M34, D35, D50, I51, T52a, N52, R53, S54, G55, G56, S57, I58, Y59, N60, E61, E62, F63, Q64, D65, R95, K96, S97, Y98, G99, Y100, Y100a, L100b, D101, and E102, in an antibody heavy chain variable domain comprising the heavy chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively; (b) measuring FVIII cofactor function-substituting activity of the variant produced in (a); and (c) selecting a variant of Emicizumab having improved FVIII cofactor function-substituting activity compared to that of Emicizumab. 